U.S. patent application number 10/729581 was filed with the patent office on 2004-10-07 for method for in vitro selection of 2'-substituted nucleic acids.
Invention is credited to Burmeister, Paula, Keefe, Anthony D., Keene, Sara Chesworth, Wilson, Charles.
Application Number | 20040197804 10/729581 |
Document ID | / |
Family ID | 32475411 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197804 |
Kind Code |
A1 |
Keefe, Anthony D. ; et
al. |
October 7, 2004 |
Method for in vitro selection of 2'-substituted nucleic acids
Abstract
Materials and methods are provided for producing aptamer
therapeutics having modified nucleotide triphosphates incorporated
into their sequence. The aptamers produced by the methods of the
invention have increased stability and half life.
Inventors: |
Keefe, Anthony D.;
(Cambridge, MA) ; Wilson, Charles; (Concord,
MA) ; Burmeister, Paula; (Cambridge, MA) ;
Keene, Sara Chesworth; (Tewksbury, MA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
32475411 |
Appl. No.: |
10/729581 |
Filed: |
December 3, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60430761 |
Dec 3, 2002 |
|
|
|
60487474 |
Jul 15, 2003 |
|
|
|
60517039 |
Nov 4, 2003 |
|
|
|
Current U.S.
Class: |
435/6.19 ;
435/91.2 |
Current CPC
Class: |
C12N 2320/13 20130101;
C12N 2310/16 20130101; C12N 2310/321 20130101; C12N 2330/30
20130101; C12N 2310/321 20130101; C12N 15/1048 20130101; C12N
9/1247 20130101; C12N 2310/322 20130101; C12N 15/115 20130101; C12N
2310/3521 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method for identifying nucleic acid ligands comprising a
modified nucleotide to a target molecule comprising: a) preparing a
transcription reaction mixture comprising a mutated polymerase, one
or more 2'-modified nucleotide triphosphates (NTPs), magnesium ions
and one or more oligonucleotide transcription templates; b)
preparing a candidate mixture of single-stranded nucleic acids by
transcribing the one or more oligonucleotide transcription
templates under conditions whereby the mutated polymerase
incorporates at least one of the one or more modified nucleotides
into each nucleic acid of said candidate mixture, wherein each
nucleic acid of said candidate mixture comprises a 2'-modified
nucleotide selected from the group consisting of a 2'-position
modified pyrimidine and a 2'-position modified purine; c)
contacting the candidate mixture with said target molecule; d)
partitioning the nucleic acids having an increased affinity to the
target molecule relative to the candidate mixture from the
remainder of the candidate mixture; and e) amplifying the increased
affinity nucleic acids, in vitro, to yield a ligand-enriched
mixture of nucleic acids, whereby nucleic acid ligands of the
target molecule are identified.
2. The method of claim 1, wherein the one or more 2'-modified
nucleotides are selected from the group consisting of 2'-OH,
2'-deoxy, 2'-O-methyl, 2'-NH.sub.2, 2'-F, and 2'-methoxy ethyl
modifications.
3. The method of claim 1, wherein the one or more 2'-modified
nucleotides are a 2'-.beta.-methyl modification.
4. The method of claim 1, wherein the one or more 2'-modified
nucleotides are a 2'-F modification.
5. The method of claim 1, wherein the mutated polymerase is a
mutated T7 RNA polymerase.
6. The method of claim 5, wherein the mutated T7 RNA polymerase
comprises a mutation at position 639 from a tyrosine residue to a
phenylalanine residue (Y639F).
7. The method of claim 5, wherein the mutated T7 RNA polymerase
comprises a mutation at position 784 from a histidine residue to an
alanine residue (H784A).
8. The method of claim 5, wherein the mutated T7 RNA polymerase
comprises a mutation at position 639 from a tyrosine residue to a
phenylalanine residue and a mutation at position 784 from a
histidine residue to an alanine residue (Y639F/H784A).
9. The method of claim 1, wherein the oligonucleotide transcription
template further comprises a leader sequence incorporated into a
fixed region at the 5' end of the oligonucleotide transcription
template.
10. The method of claim 9, wherein the leader sequence comprises an
all-purine leader sequence.
11. The method of claim 10, wherein the all-purine leader sequence
has a length selected from the group consisting of at least 6
nucleotides long; at least 8 nucleotides long; at least 10
nucleotides long; at least 12 nucleotides long; and at least 14
nucleotides long.
12. The method of claim 1, wherein the transcription reaction
mixture further comprises manganese ions.
13. The method of claim 12, wherein the concentration of magnesium
ions is between 3.0 and 3.5 times greater than the concentration of
manganese ions.
14. The method of claim 1, wherein each NTP is present at a
concentration of 0.5 mM, the concentration of magnesium ions is 5.0
mM, and the concentration of manganese ions is 1.5 mM.
15. The method of claim 1, wherein each NTP is present at a
concentration of 1.0 mM, the concentration of magnesium ions is 6.5
mM, and the concentration of manganese ions is 2.0 mM.
16. The method of claim 1, wherein each NTP is present at a
concentration of 2.0 mM, the concentration of magnesium ions is 9.6
mM, and the concentration of manganese ions is 2.9 mM.
17. The method of claim 1, wherein the transcription reaction
mixture further comprises 2'-OH GTP.
18. The method of claim 1, wherein the transcription reaction
mixture further comprises a polyalkylene glycol.
19. The method of claim 18, wherein the polyalkylene glycol is
polyethylene glycol (PEG).
20. The method of claim 1, wherein the transcription reaction
mixture further comprises GMP.
21. The method of claim 1 further comprising f) repeating steps d)
and e).
22. A nucleic acid ligand to thrombin identified according to the
method of claim 1.
23. A nucleic acid ligand to vascular endothelial growth factor
(VEGF) identified according to the method of claim 1.
24. A nucleic acid ligand to IgE identified according to the method
of claim 1.
25. A nucleic acid ligand to IL-23 identified according to the
method of claim 1.
26. A nucleic acid ligand to platelet-derived growth factor-BB
(PDGF-BB) identified according to the method of claim 1.
27. The method of claim 1, wherein the 2' modified nucleotide
triphosphates comprise a mixture of 2'-OH adenosine triphosphate
(ATP), 2'-OH guanosine triphosphate (GTP), 2'-O-methyl cytidine
triphosphate (CTP) and 2'-O-methyl uridine triphosphate (UTP).
28. The method of claim 1, wherein the 2' modified nucleotide
triphosphates comprise a mixture of 2'-deoxy purine nucleotide
triphosphates and 2'-O-methylpyrimidine nucleotide
triphosphates.
29. The method of claim 1, wherein the 2' modified nucleotide
triphosphates comprise a mixture of 2'-O-methyl adenosine
triphosphate (ATP), 2'-OH guanosine triphosphate (GTP), 2'-O-methyl
cytidine triphosphate (CTP) and 2'-O-methyl uridine triphosphate
(UTP).
30. The method of claim 1, wherein the 2' modified nucleotide
triphosphates comprise a mixture of 2'-O-methyl adenosine
triphosphate (ATP), 2'-O-methyl cytidine triphosphate (CTP) and
2'-O-methyl uridine triphosphate (UTP), 2'-O-methyl guanosine
triphosphate (GTP) and deoxy guanosine triphosphate (GTP), wherein
the deoxy guanosine triphosphate comprises a maximum of 10% of the
total guanosine triphosphate population.
31. The method of claim 1, wherein the 2' modified nucleotide
triphosphates comprise a mixture of 2'-O-methyl adenosine
triphosphate (ATP), 2'-F guanosine triphosphate (GTP), 2'-O-methyl
cytidine triphosphate (CTP) and 2'-O-methyl uridine triphosphate
(UTP).
32. The method of claim 1, wherein the 2' modified nucleotide
triphosphates comprise a mixture of 2'-deoxy adenosine triphosphate
(ATP), 2'-O-methyl guanosine triphosphate (GTP), 2'-O-methyl
cytidine triphosphate (CTP) and 2'-O-methyl uridine triphosphate
(UTP).
33. A method of preparing a nucleic acid comprising one or more
modified nucleotides comprising: (a) preparing a transcription
reaction mixture comprising a mutated polymerase, one or more
2'-modified nucleotide triphosphates (NTPs), magnesium ions and one
or more oligonucleotide transcription templates; and (b) contacting
the one or more oligonucleotide transcription templates with the
mutated polymerase under conditions whereby the mutated polymerase
incorporates the one or more 2'-modified nucleotides into a nucleic
acid transcription product.
34. The method of claim 33, wherein the one or more 2'-modified
nucleotides are selected from the group consisting of 2'-OH,
2'-deoxy, 2'-O-methyl, 2'-NH.sub.2, 2'-F, and 2'-methoxy ethyl
modifications.
35. The method of claim 33, wherein the one or more 2'-modified
nucleotides are a 2'-O-methyl modification.
36. The method of claim 33, wherein the one or more 2'-modified
nucleotides are a 2'-F modification.
37. The method of claim 33, wherein the mutated polymerase is a
mutated T7 RNA polymerase.
38. The method of claim 37, wherein the mutated T7 RNA polymerase
comprises a mutation at position 639 from a tyrosine residue to a
phenylalanine residue (Y639F).
39. The method of claim 37, wherein the mutated T7 RNA polymerase
comprises a mutation at position 784 from a histidine residue to an
alanine residue (H784A).
40. The method of claim 37, wherein the mutated T7 RNA polymerase
comprises a mutation at position 639 from a tyrosine residue to a
phenylalanine residue and a mutation at position 784 from a
histidine residue to an alanine residue (Y639F/H784A).
41. The method of claim 33, wherein the oligonucleotide
transcription template further comprises a leader sequence
incorporated into a fixed region at the 5' end of the
oligonucleotide transcription template.
42. The method of claim 41, wherein the leader sequence comprises
an all-purine leader sequence.
43. The method of claim 42, wherein the all-purine leader sequence
has a length selected from the group consisting of at least 6
nucleotides long; at least 8 nucleotides long; at least 10
nucleotides long; at least 12 nucleotides long; and at least 14
nucleotides long.
44. The method of claim 33, wherein the transcription reaction
mixture further comprises manganese ions.
45. The method of claim 44, wherein the concentration of magnesium
ions is between 3.0 and 3.5 times greater than the concentration of
manganese ions.
46. The method of claim 33, wherein each NTP is present at a
concentration of 0.5 mM each, the concentration of magnesium ions
is 5.0 mM, and the concentration of manganese ions is 1.5 mM.
47. The method of claim 33, wherein each NTP is present at a
concentration of 1.0 mM each, the concentration of magnesium ions
is 6.5 mM, and the concentration of manganese ions is 2.0 mM.
48. The method of claim 33, wherein each NTP is present at a
concentration of 2.0 mM each, the concentration of magnesium ions
is 9.6 mM, and the concentration of manganese ions is 2.9 mM.
49. The method of claim 33, wherein the transcription reaction
mixture further comprises 2'-OH GTP.
50. The method of claim 33, wherein the transcription reaction
mixture further comprises a polyalkylene glycol.
51. The method of claim 50, wherein the polyalkylene glycol is
polyethylene glycol (PEG).
52. The method of claim 33, wherein the transcription reaction
mixture further comprises GMP.
53. An aptamer composition comprising a sequence where
substantially all adenosine nucleotides are 2'-OH adenosine,
substantially all guanosine nucleotides are 2'-OH guanosine,
substantially all cytidine nucleotides are 2'-O-methyl cytidine,
and substantially all uridine nucleotides are 2'-O-methyl
uridine.
54. The aptamer composition of claim 53 wherein said aptamer
comprises a sequence composition where at least 80% of all
adenosine nucleotides are 2'-OH adenosine, at least 80% of all
guanosine nucleotides are 2'-OH guanosine, at least 80% of all
cytidine nucleotides are 2'-O-methyl cytidine and at least 80% of
all uridine nucleotides are 2'-O-methyl uridine.
55. The aptamer composition of claim 53 wherein said aptamer
comprises a sequence composition where at least 90% of all
adenosine nucleotides are 2'-OH adenosine, at least 90% of all
guanosine nucleotides are 2'-OH guanosine, at least 90% of all
cytidine nucleotides are 2'-O-methyl cytidine and at least 90% of
all uridine nucleotides are 2'-O-methyl uridine.
56. The aptamer composition of claim 53 wherein said aptamer
comprises a sequence composition where 100% of all adenosine
nucleotides are 2'-OH adenosine, at 100% of all guanosine
nucleotides are 2'-OH guanosine, 100% of all cytidine nucleotides
are 2'-O-methyl cytidine and 100% of all uridine nucleotides are
2'-O-methyl uridine.
57. An aptamer composition comprising a sequence where
substantially all purine nucleotides are 2'-deoxy purines and
substantially all pyrimidine nucleotides are 2'-O-methyl
pyrimidines.
58. The aptamer composition of claim 57 wherein said aptamer
comprises a sequence composition where at least 80% of all purine
nucleotides are 2'-deoxy purines and at least 80% of all pyrimidine
nucleotides are 2'-O-methylpyrimidines.
59. The aptamer composition of claim 57 wherein said aptamer
comprises a sequence composition where at least 90% of all purine
nucleotides are 2'-deoxy purines and at least 90% of all pyrimidine
nucleotides are 2'-O-methylpyrimidines.
60. The aptamer composition of claim 57 wherein said aptamer
comprises a sequence composition where 100% of all purine
nucleotides are 2'-deoxy purines and 100% of all pyrimidine
nucleotides are 2'-O-methyl pyrimidines
61. An aptamer composition comprising a sequence composition where
substantially all guanosine nucleotides are 2'-OH guanosine,
substantially all cytidine nucleotides are 2'-O-methyl cytidine,
substantially all uridine nucleotides are 2'-O-methyl uridine, and
substantially all adenosine nucleotides are 2'-O-methyl
adenosine.
62. The aptamer composition of claim 61 wherein said aptamer
comprises a sequence composition where at least 80% of all
guanosine nucleotides are 2'-OH guanosine, at least 80% of all
cytidine nucleotides are 2'-O-methyl cytidine, at least 80% of all
uridine nucleotides are 2'-O-methyl uridine, and at least 80% of
all adenosine nucleotides are 2'-O-methyl adenosine.
63. The aptamer composition of claim 61 wherein said aptamer
comprises a sequence composition where at least 90% of all
guanosine nucleotides are 2'-OH guanosine, at least 90% of all
cytidine nucleotides are 2'-O-methyl cytidine, at least 90% of all
uridine nucleotides are 2'-O-methyl uridine, and at least 90% of
all adenosine nucleotides are 2'-O-methyl adenosine.
64. The aptamer composition of claim 61 wherein said aptamer
comprises a sequence composition where 100% of all guanosine
nucleotides are 2'-OH guanosine, 100% of all cytidine nucleotides
are 2'-O-methyl cytidine, 100% of all uridine nucleotides are
2'-O-methyl uridine, and 100% of all adenosine nucleotides are
2'-O-methyl adenosine.
65. An aptamer composition comprising a sequence where
substantially all adenosine nucleotides are 2'-O-methyl adenosine,
substantially all cytidine nucleotides are 2'-O-methyl cytidine,
substantially all guanosine nucleotides are 2'-O-methyl guanosine
or deoxy guanosine, substantially all uridine nucleotides are
2'-O-methyl uridine, wherein less than about 10% of the guanosine
nucleotides are deoxy guanosine.
66. The aptamer composition of claim 65 wherein said aptamer
comprises a sequence composition where at least 80% of all
adenosine nucleotides are 2'-O-methyl adenosine, at least 80% of
all cytidine nucleotides are 2'-O-methyl cytidine, at least 80% of
all guanosine nucleotides are 2'-O-methyl guanosine, at least 80%
of all uridine nucleotides are 2'-O-methyl uridine, and no more
than about 10% of all guanosine nucleotides are deoxy
guanosine.
67. The aptamer composition of claim 65 wherein said aptamer
comprises a sequence composition where at least 90% of all
adenosine nucleotides are 2'-O-methyl adenosine, at least 90% of
all cytidine nucleotides are 2'-O-methyl cytidine, at least 90% of
all guanosine nucleotides are 2'-O-methyl guanosine, at least 90%
of all uridine nucleotides are 2'-O-methyl uridine, and no more
than about 10% of all guanosine nucleotides are deoxy
guanosine.
68. The aptamer composition of claim 65 wherein said aptamer
comprises a sequence composition where 100% of all adenosine
nucleotides are 2'-O-methyl adenosine, 100% of all cytidine
nucleotides are 2'-O-methyl cytidine, 90% of all guanosine
nucleotides are 2'-O-methyl guanosine, and 100% of all uridine
nucleotides are 2'-O-methyl uridine and no more than about 10% of
all guanosine nucleotides are deoxy guanosine.
69. An aptamer composition comprising a sequence where
substantially all adenosine nucleotides are 2'-O-methyl adenosine,
substantially all uridine nucleotides are 2'-O-methyl uridine,
substantially all cytidine nucleotides are 2'-O-methyl cytidine,
and substantially all guanosine nucleotides are 2'-F guanosine
sequence.
70. The aptamer composition of claim 69 wherein said aptamer
comprises a sequence composition where at least 80% of all
adenosine nucleotides are 2'-O-methyl adenosine, at least 80% of
all uridine nucleotides are 2'-O-methyl uridine, at least 80% of
all cytidine nucleotides are 2'-O-methyl cytidine, and at least 80%
of all guanosine nucleotides are 2'-F guanosine.
71. The aptamer composition of claim 69 wherein said aptamer
comprises a sequence composition where at least 90% of all
adenosine nucleotides are 2'-O-methyl adenosine, at least 90% of
all uridine nucleotides are 2'-O-methyl uridine, at least 90% of
all cytidine nucleotides are 2'-O-methyl cytidine, and at least 90%
of all guanosine nucleotides are 2'-F guanosine
72. The aptamer composition of claim 69 wherein said aptamer
comprises a sequence composition where 100% of all adenosine
nucleotides are 2'-O-methyl adenosine, 100% of all uridine
nucleotides are 2'-O-methyl uridine, 100% of all cytidine
nucleotides are 2'-O-methyl cytidine, and 100% of all guanosine
nucleotides are 2'-F guanosine.
73. An aptamer composition comprising a sequence where
substantially all adenosine nucleotides are 2'-deoxy adenosine,
substantially all cytidine nucleotides are 2'-O-methyl cytidine,
substantially all guanosine nucleotides are 2'-O-methyl guanosine,
and substantially all uridine nucleotides are 2'-O-methyl
uridine.
74. The aptamer composition of claim 73 wherein said aptamer
comprises a sequence composition where at least 80% of all
adenosine nucleotides are 2'-deoxy adenosine, at least 80% of all
cytidine nucleotides are 2'-O-methyl cytidine, at least 80% of all
guanosine nucleotides are 2'-O-methyl guanosine, and at least 80%
of all uridine nucleotides are 2'-O-methyl uridine.
75. The aptamer composition of claim 73 wherein said aptamer
comprises a sequence composition where at least 90% of all
adenosine nucleotides are 2'-deoxy adenosine, at least 90% of all
cytidine nucleotides are 2'-O-methyl cytidine, at least 90% of all
guanosine nucleotides are 2'-O-methyl guanosine, and at least 90%
of all uridine nucleotides are 2'-O-methyl uridine.
76. The aptamer composition of claim 73 wherein said aptamer
comprises a sequence composition where 100% of all adenosine
nucleotides are 2'-deoxy adenosine, 100% of all cytidine
nucleotides are 2'-O-methyl cytidine, 100% of all guanosine
nucleotides are 2'-O-methyl guanosine, and 100% of all uridine
nucleotides are 2'-O-methyl uridine.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to and claims priority to U.S.
Provisional Patent Application Serial No. 60/430,761, filed Dec. 3,
2002, U.S. Provisional Patent Application Serial No. 60/487,474,
filed Jul. 15, 2003, and U.S. Provisional Patent Application Serial
No. 60/517,039, filed Nov. 4, 2003, each of which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to the field of nucleic
acids and more particularly to aptamers, and methods for selecting
aptamers, incorporating modified nucleotides. The invention further
relates to materials and methods for enzymatically producing pools
of randomized oligonucleotides having modified nucleotides from
which, e.g., aptamers to a specific target can be selected.
BACKGROUND OF THE INVENTION
[0003] Aptamers are nucleic acid molecules having specific binding
affinity to molecules through interactions other than classic
Watson-Crick base pairing.
[0004] Aptamers, like peptides generated by phage display or
monoclonal antibodies (MAbs), are capable of specifically binding
to selected targets and, through binding, block their targets'
ability to function. Created by an in vitro selection process from
pools of random sequence oligonucleotides (FIG. 1), aptamers have
been generated for over 100 proteins including growth factors,
transcription factors, enzymes, immunoglobulins, and receptors. A
typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its
target with sub-nanomolar affinity, and discriminates against
closely related targets (e.g., will typically not bind other
proteins from the same gene family). A series of structural studies
have shown that aptamers are capable of using the same types of
binding interactions (hydrogen bonding, electrostatic
complementarity, hydrophobic contacts, steric exclusion, etc) that
drive affinity and specificity in antibody-antigen complexes.
[0005] Aptamers have a number of desirable characteristics for use
as therapeutics (and diagnostics) including high specificity and
affinity, biological efficacy, and excellent pharmacokinetic
properties. In addition, they offer specific competitive advantages
over antibodies and other protein biologics, for example:
[0006] 1) Speed and control. Aptamers are produced by an entirely
in vitro process, allowing for the rapid generation of initial
(therapeutic) leads. In vitro selection allows the specificity and
affinity of the aptarner to be tightly controlled and allows the
generation of leads against both toxic and non-immunogenic
targets.
[0007] 2) Toxicity and Immunogenicity. Aptamers as a class have
demonstrated little or no toxicity or immunogenicity. In chronic
dosing of rats or woodchucks with high levels of aptamer (10 mg/kg
daily for 90 days), no toxicity is observed by any clinical,
cellular, or biochemical measure. Whereas the efficacy of many
monoclonal antibodies can be severely limited by immune response to
antibodies themselves, it is extremely difficult to elicit
antibodies to aptamers (most likely because aptamers cannot be
presented by T-cells via the MHC and the immune response is
generally trained not to recognize nucleic acid fragments).
[0008] 3) Administration. Whereas all currently approved antibody
therapeutics are administered by intravenous infusion (typically
over 2-4 hours), aptamers can be administered by subcutaneous
injection. This difference is primarily due to the comparatively
low solubility and thus large volumes necessary for most
therapeutic MAbs. With good solubility (>150 mg/ml) and
comparatively low molecular weight (aptarner: 10-50 kDa; antibody:
150 kDa), a weekly dose of aptarner may be delivered by injection
in a volume of less than 0.5 ml. Aptamer bioavailability via
subcutaneous administration is >80% in monkey studies (Tucker et
al., J. Chromatography B. 732: 203-12, 1999). In addition, the
small size of aptamers allows them to penetrate into areas of
conformational constrictions that do not allow for antibodies or
antibody fragments to penetrate, presenting yet another advantage
of aptamer-based therapeutics or prophylaxis.
[0009] 4) Scalability and cost. Therapeutic aptamers are chemically
synthesized and consequently can be readily scaled as needed to
meet production demand. Whereas difficulties in scaling production
are currently limiting the availability of some biologics and the
capital cost of a large-scale protein production plant is enormous,
a single large-scale synthesizer can produce upwards of 100 kg
oligonucleotide per year and requires a relatively modest initial
investment. The current cost of goods for aptamer synthesis at the
kilogram scale is estimated at $500/g, comparable to that for
highly optimized antibodies. Continuing improvements in process
development are expected to lower the cost of goods to <$100/g
in five years.
[0010] 5) Stability. Therapeutic aptamers are chemically robust.
They are intrinsically adapted to regain activity following
exposure to heat, denaturants, etc. and can be stored for extended
periods (>1 yr) at room temperature as lyophilized powders. In
contrast, antibodies must be stored refrigerated.
[0011] Given the advantages of aptamers as therapeutic agents, it
would be beneficial to have materials and methods to prolong or
increase the stability of aptamer therapeutics in vivo. The present
invention provides materials and methods to meet these and other
needs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic representation of the in vitro aptamer
selection (SELEX.TM.) process from pools of random sequence
oligonucleotides.
[0013] FIG. 2 shows a 2'-O-methyl (2'-OMe) modified nucleotide,
where "B" is a purine or pyrimidine base.
[0014] FIG. 3A is a graph of VEGF-binding by three 2'-OMe VEGF
aptamers: ARC224, ARC245 and ARC259; FIG. 3B shows the sequences
and putative secondary structures of these aptamers.
[0015] FIG. 4 is a graph of the VEGF-binding by various 2'-OH G
variants of ARC224 and ARC225
[0016] FIG. 5 is a graph of ARC224 binding to VEGF in HUVEC.
[0017] FIG. 6 is a graph of ARC224 binding to VEGF before and after
autoclaving, in the presence or absence of EDTA.
[0018] FIGS. 7A and 7B are graphs of the stability of ARC224 and
ARC226, respectively, when incubated at 37.degree. C. in rat
plasma.
[0019] FIG. 8 is a graph of dRmY SELEX.TM. Round 6 sequences
binding to IgE.
[0020] FIG. 9 is a graph of dRmY SELEX.TM. Round 6 sequences
binding to thrombin.
[0021] FIG. 10 is a graph of dRmY SELEX.TM. Round 6 sequences
binding to VEGF.
[0022] FIG. 11A is a degradation plot of an all 2'-OMe
oligonucleotide with 3'-idT, in 95% rat plasma (citrated) at
37.degree. C., and FIG. 11B is a degradation plot of the
corresponding dRmY oligonucleotide in 95% rat plasma at 37.degree.
C.
[0023] FIG. 12 is a graph of rGmH h-IgE binding clones (Round
6).
[0024] FIG. 13A is a graph of round 12 pools for rRmY pool PDGF-BB
selection, and FIG. 13B is a graph of Round 10 pools for rGmH pool
PDGF-BB selection.
[0025] FIG. 14 is a graph of dRmY SELEX.TM. Round 6, 7, 8 and
unselected sequences binding to IL-23.
[0026] FIG. 15 is a graph of dRmY SELEX.TM. Round 6, 7 and
unselected sequences binding to PDGF-BB.
SUMMARY OF THE INVENTION
[0027] The present invention provides materials and methods to
produce oligonucleotides of increased stability by transcription
under the conditions specified herein which promote the
incorporation of modified nucleotides into the oligonucleotide.
These modified oligonucleotides can be, for example, aptamers,
antisense molecules, RNAi molecules, siRNA molecules, or ribozymes.
Preferably, the oligonucleotide is an aptamer.
[0028] In one embodiment, the present invention provides an
improved SELEX.TM. method ("2"-OMe SELEX.TM.") that uses randomized
pools of oligonucleotides incorporating modified nucleotides from
which aptamers to a specific target can be selected.
[0029] In one embodiment, the present invention provides methods
that use modified enzymes to incorporate modified nucleotides into
oligonucleotides under a given set of transcription conditions.
[0030] In one embodiment, the present invention provides methods
that use a mutated polymerase. In one embodiment, the mutated
polymerase is a T7 RNA polymerase. In one embodiment, a T7 RNA
polymerase modified by having a mutation at position 639 (from a
tyrosine residue to a phenylalanine residue "Y639F") and at
position 784 (from a histidine residue to an alanine residue
"H784A") is used in various transcription reaction conditions which
result in the incorporation of modified nucleotides into the
oligonucleotides of the invention.
[0031] In another embodiment, a T7 RNA polymerase modified with a
mutation at position 639 (from a tyrosine residue to a
phenylalanine residue) is used in various transcription reaction
conditions which result in the incorporation of modified
nucleotides into the oligonucleotides of the invention.
[0032] In another embodiment, a T7 RNA polymerase modified with a
mutation at position 784 (from a histidine residue to an alanine
residue) is used in various transcription reaction conditions which
result in the incorporation of modified nucleotides into the
aptamers of the invention.
[0033] In one embodiment, the present invention provides various
transcription reaction mixtures that increase the incorporation of
modified nucleotides by the modified enzymes of the invention.
[0034] In one embodiment, manganese ions are added to the
transcription reaction mixture to increase the incorporation of
modified nucleotides by the modified enzymes of the invention.
[0035] In another embodiment, 2'-OH GTP is added to the
transcription mixture to increase the incorporation of modified
nucleotides by the modified enzymes of the invention.
[0036] In another embodiment, polyethylene glycol, PEG, is added to
the transcription mixture to increase the incorporation of modified
nucleotides by the modified enzymes of the invention.
[0037] In another embodiment, GMP (or any substituted guanosine) is
added to the transcription mixture to increase the incorporation of
modified nucleotides by the modified enzymes of the invention.
[0038] In one embodiment, a leader sequence incorporated into the
5' end of the fixed region (preferably 20-25 nucleotides in length)
at the 5' end of a template oligonucleotide is used to increase the
incorporation of modified nucleotides by the modified enzymes of
the invention. Preferably, the leader sequence is greater than
about 10 nucleotides in length.
[0039] In one embodiment, a leader sequence that is composed of up
to 100% (inclusive) purine nucleotides is used.
[0040] In another embodiment, a leader sequence at least 6
nucleotides long that is composed of up to 100% (inclusive) purine
nucleotides is used.
[0041] In another embodiment, a leader sequence at least 8
nucleotides long that is composed of up to 100% (inclusive) purine
nucleotides is used.
[0042] In another embodiment, a leader sequence at least 10
nucleotides long that is composed of up to 100% (inclusive) purine
nucleotides is used.
[0043] In another embodiment, a leader sequence at least 12
nucleotides long that is composed of up to 100% (inclusive) purine
nucleotides is used.
[0044] In another embodiment, a leader sequence at least 14
nucleotides long that is composed of up to 100% (inclusive) purine
nucleotides is used.
[0045] In one embodiment, the present invention provides aptamer
therapeutics having modified nucleotides incorporated into their
sequence.
[0046] In one embodiment, the present invention provides for the
use of aptamer therapeutics having modified nucleotides
incorporated into their sequence.
[0047] In one embodiment, the present invention provides various
compositions of nucleotides for transcription for the selection of
aptamers with the SELEX.TM. process. In one embodiment, the present
invention provides combinations of 2'-OH, 2'-F, 2'-deoxy, and
2'-OMe modifications of the ATP, GTP, CTP, TTP, and UTP
nucleotides. In another embodiment, the present invention provides
combinations of 2'-OH, 2'-F, 2'-deoxy, 2'-OMe, 2'-NH.sub.2, and
2'-methoxyethyl modifications of the ATP, GTP, CTP, TTP, and UTP
nucleotides. In one embodiment, the present invention provides 5
combinations of 2'-OH, 2'-F, 2'-deoxy, 2'-OMe, 2'-NH.sub.2, and
2'-methoxyethyl modifications the ATP, GTP, CTP, TTP, and UTP
nucleotides.
[0048] The invention relates to a method for identifying nucleic
acid ligands to a target molecule, where the ligands include
modified nucleotides, by: a) preparing a transcription reaction
mixture comprising a mutated polymerase, one or more 2'-modified
nucleotide triphosphates (NTPs), magnesium ions and one or more
oligonucleotide transcription templates; b) preparing a candidate
mixture of single-stranded nucleic acids by transcribing the one or
more oligonucleotide transcription templates under conditions
whereby the mutated polymerase incorporates at least one of the one
or more modified nucleotides into each nucleic acid of the
candidate mixture, wherein each nucleic acid of the candidate
mixture comprises a 2'-modified nucleotide selected from the group
consisting of a 2'-position modified pyrimidine and a 2'-position
modified purine; c) contacting the candidate mixture with the
target molecule; d) partitioning the nucleic acids having an
increased affinity to the target molecule relative to the candidate
mixture from the remainder of the candidate mixture; and e)
amplifying the increased affinity nucleic acids, in vitro, to yield
a ligand-enriched mixture of nucleic acids.
[0049] The 2'-position modified pyrimidines and 2'-position
modified purines include 2'-OH, 2'-deoxy, 2'-O-methyl, 2'-NH.sub.2,
2'-F, and 2'-methoxy ethyl modifications. Preferably, the
2'-modified nucleotides are 2'-O-methyl or 2'-F nucleotides.
[0050] In some embodiments, the mutated polymerase is a mutated T7
RNA polymerase, such as a T7 RNA polymerase having a mutation at
position 639 from a tyrosine residue to a phenylalanine residue
(Y639F); a T7 RNA polymerase having a mutation at position 784 from
a histidine residue to an alanine residue (H784A); a T7 RNA
polymerase having a mutation at position 639 from a tyrosine
residue to a phenylalanine residue and a mutation at position 784
from a histidine residue to an alanine residue (Y639F/H784A).
[0051] In some embodiments, the oligonucleotide transcription
template includes a leader sequence incorporated into the 5' end of
a fixed region at the 5' end of the oligonucleotide transcription
template. The leader sequence, for example, is an all-purine leader
sequence. The leader sequence, for example, can be at least 6
nucleotides long; at least 8 nucleotides long; at least 10
nucleotides long; at least 12 nucleotides long; or at least 14
nucleotides long.
[0052] In some embodiments, the transcription reaction mixture also
includes manganese ions. For example, the concentration of
magnesium ions is between 3.0 and 3.5 times greater than the
concentration of manganese ions.
[0053] In some embodiments of the transcription reaction mixture,
each NTP is present at a concentration of 0.5 mM, the concentration
of magnesium ions is 5.0 mM, and the concentration of manganese
ions is 1.5 mM. In other embodiments of the transcription reaction
mixture each NTP is present at a concentration of 1.0 mM, the
concentration of magnesium ions is 6.5 mM, and the concentration of
manganese ions is 2.0 mM. In other embodiments of the transcription
reaction mixture each NTP is present at a concentration of 2.0 mM,
the concentration of magnesium ions is 9.6 mM, and the
concentration of manganese ions is 2.9 mM.
[0054] In some embodiments, the transcription reaction mixture also
includes 2'-OH GTP.
[0055] In some embodiments, the transcription reaction mixture also
includes a polyalkylene glycol. The polyalkylene glycol can be,
e.g., polyethylene glycol (PEG).
[0056] In some embodiments, the transcription reaction mixture also
includes GMP.
[0057] In some embodiments, the method for identifying nucleic acid
ligands to a target molecule further includes repeating steps d)
partitioning the nucleic acids having an increased affinity to the
target molecule relative to the candidate mixture from the
remainder of the candidate mixture; and e) amplifying the increased
affinity nucleic acids, in vitro, to yield a ligand-enriched
mixture of nucleic acids.
[0058] In some aspects, the invention relates to a nucleic acid
ligand to thrombin which was identified according to the method of
the invention.
[0059] In some aspects, the invention relates to a nucleic acid
ligand to vascular endothelial growth factor (VEGF) which was
identified according to the method of the invention.
[0060] In some aspects, the invention relates to a nucleic acid
ligand to IgE which was identified according to the method of the
invention.
[0061] In some aspects, the invention relates to a nucleic acid
ligand to IL-23 which was identified according to the method of the
invention.
[0062] In some aspects, the invention relates to a nucleic acid
ligand to platelet-derived growth factor-BB (PDGF-BB) which was
identified according to the method of the invention.
[0063] In some embodiments, the transcription reaction mixture
includes 2'-OH adenosine triphosphate (ATP), 2'-OH guanosine
triphosphate (GTP), 2'-O-methyl cytidine triphosphate (CTP) and
2'-O-methyl uridine triphosphate (UTP).
[0064] In some embodiments, the transcription reaction mixture
includes 2'-deoxy purine nucleotide triphosphates and
2'-O-methylpyrimidine nucleotide triphosphates.
[0065] In some embodiments, the transcription reaction mixture
includes 2'-O-methyl adenosine triphosphate (ATP), 2'-OH guanosine
triphosphate (GTP), 2'-O-methyl cytidine triphosphate (CTP) and
2'-O-methyl uridine triphosphate (UTP).
[0066] In some embodiments, the transcription reaction mixture
includes 2'-O-methyl adenosine triphosphate (ATP), 2'-O-methyl
cytidine triphosphate (CTP) and 2'-O-methyl uridine triphosphate
(UTP), 2'-O-methyl guanosine triphosphate (GTP) and deoxy guanosine
triphosphate (GTP), wherein the deoxy guanosine triphosphate
comprises a maximum of 10% of the total guanosine triphosphate
population.
[0067] In some embodiments, the transcription reaction mixture
includes 2'-O-methyl adenosine triphosphate (ATP), 2'-F guanosine
triphosphate (GTP), 2'-O-methyl cytidine triphosphate (CTP) and
2'-O-methyl uridine triphosphate (UTP).
[0068] In some embodiments, the transcription reaction mixture
includes 2'-deoxy adenosine triphosphate (ATP), 2'-O-methyl
guanosine triphosphate (GTP), 2'-O-methyl cytidine triphosphate
(CTP) and 2'-O-methyl uridine triphosphate (UTP).
[0069] The invention also relates to a method of preparing a
nucleic acid comprising one or more modified nucleotides by:
preparing a transcription reaction mixture comprising a mutated
polymerase, one or more 2'-modified nucleotide triphosphates
(NTPs), magnesium ions and one or more oligonucleotide
transcription templates; and contacting the one or more
oligonucleotide transcription templates with the mutated polymerase
under conditions whereby the mutated polymerase incorporates the
one or more 2'-modified nucleotides into a nucleic acid
transcription product.
[0070] 2'-position modified pyrimidines and 2'-position modified
purines include 2'-OH, 2'-deoxy, 2'-O-methyl, 2'-NH.sub.2, 2'-F,
and 2'-methoxy ethyl modifications. Preferably, the 2'-modified
nucleotides are 2'-O-methyl or 2'-F nucleotides.
[0071] In some embodiments, the mutated polymerase is a mutated T7
RNA polymerase, such as a T7 RNA polymerase having a mutation at
position 639 from a tyrosine residue to a phenylalanine residue
(Y639F); a T7 RNA polymerase having a mutation at position 784 from
a histidine residue to an alanine residue (H784A); a T7 RNA
polymerase having a mutation at position 639 from a tyrosine
residue to a phenylalanine residue and a mutation at position 784
from a histidine residue to an alanine residue (Y639F/H784A).
[0072] In some embodiments, the oligonucleotide transcription
template includes a leader sequence incorporated into the 5' end of
a fixed region at the 5' end of the oligonucleotide transcription
template. The leader sequence, for example, is an all-purine leader
sequence. The leader sequence, for example, can be at least 6
nucleotides long; at least 8 nucleotides long; at least 10
nucleotides long; at least 12 nucleotides long; or at least 14
nucleotides long.
[0073] In some embodiments, the transcription reaction mixture also
includes manganese ions. For example, the concentration of
magnesium ions is between 3.0 and 3.5 times greater than the
concentration of manganese ions.
[0074] In some embodiments of the transcription reaction mixture,
each NTP is present at a concentration of 0.5 mM, the concentration
of magnesium ions is 5.0 mM, and the concentration of manganese
ions is 1.5 mM. In other embodiments of the transcription reaction
mixture each NTP is present at a concentration of 1.0 mM, the
concentration of magnesium ions is 6.5 mM, and the concentration of
manganese ions is 2.0 mM. In other embodiments of the transcription
reaction mixture each NTP is present at a concentration of 2.0 mM,
the concentration of magnesium ions is 9.6 mM, and the
concentration of manganese ions is 2.9 mM.
[0075] In some embodiments, the transcription reaction mixture also
includes 2'-OH GTP.
[0076] In some embodiments, the transcription reaction mixture also
includes a polyalkylene glycol. The polyalkylene glycol can be,
e.g., polyethylene glycol (PEG).
[0077] In some embodiments, the transcription reaction mixture also
includes GMP.
[0078] The invention also relates to an aptamer composition
comprising a sequence where substantially all adenosine nucleotides
are 2'-OH adenosine, substantially all guanosine nucleotides are
2'-OH guanosine, substantially all cytidine nucleotides are
2'-O-methyl cytidine, and substantially all uridine nucleotides are
2'-O-methyl uridine. In one embodiment, the aptamer has a sequence
composition where at least 80% of all adenosine nucleotides are
2'-OH adenosine, at least 80% of all guanosine nucleotides are
2'-OH guanosine, at least 80% of all cytidine nucleotides are
2'-O-methyl cytidine and at least 80% of all uridine nucleotides
are 2'-O-methyl uridine. In another embodiment, the aptamer has a
sequence composition where at least 90% of all adenosine
nucleotides are 2'-OH adenosine, at least 90% of all guanosine
nucleotides are 2'-OH guanosine, at least 90% of all cytidine
nucleotides are 2'-O-methyl cytidine and at least 90% of all
uridine nucleotides are 2'-O-methyl uridine. In another embodiment,
the aptamer has a sequence composition where 100% of all adenosine
nucleotides are 2'-OH adenosine, at 100% of all guanosine
nucleotides are 2'-OH guanosine, 100% of all cytidine nucleotides
are 2'-O-methyl cytidine and 100% of all uridine nucleotides are
2'-O-methyl uridine.
[0079] The invention also relates to an aptamer composition
comprising a sequence where substantially all purine nucleotides
are 2'-deoxy purines and substantially all pyrimidine nucleotides
are 2'-O-methylpyrimidines. In one embodiment, the aptamer has a
sequence composition where at least 80% of all purine nucleotides
are 2'-deoxy purines and at least 80% of all pyrimidine nucleotides
are 2'-O-methylpyrimidines. In another embodiment, the aptamer has
a sequence composition where at least 90% of all purine nucleotides
are 2'-deoxy purines and at least 90% of all pyrimidine nucleotides
are 2'-O-methylpyrimidines. In another embodiment, the aptamer has
a sequence composition where 100% of all purine nucleotides are
2'-deoxy purines and 100% of all pyrimidine nucleotides are
2'-O-methylpyrimidines.
[0080] The invention also relates to an aptamer composition
comprising a sequence where substantially all guanosine nucleotides
are 2'-OH guanosine, substantially all cytidine nucleotides are
2'-O-methyl cytidine, substantially all uridine nucleotides are
2'-O-methyl uridine, and substantially all adenosine nucleotides
are 2'-O-methyl adenosine. In one embodiment, the aptamer has a
sequence composition where at least 80% of all guanosine
nucleotides are 2'-OH guanosine, at least 80% of all cytidine
nucleotides are 2'-O-methyl cytidine, at least 80% of all uridine
nucleotides are 2'-O-methyl uridine, and at least 80% of all
adenosine nucleotides are 2'-O-methyl adenosine. In another
embodiment, the aptamer has a sequence composition where at least
90% of all guanosine nucleotides are 2'-OH guanosine, at least 90%
of all cytidine nucleotides are 2'-O-methyl cytidine, at least 90%
of all uridine nucleotides are 2'-O-methyl uridine, and at least
90% of all adenosine nucleotides are 2'-O-methyl adenosine. In
another embodiment, the aptamer has a sequence composition where
100% of all guanosine nucleotides are 2'-OH guanosine, 100% of all
cytidine nucleotides are 2'-O-methyl cytidine, 100% of all uridine
nucleotides are 2'-O-methyl uridine, and 100% of all adenosine
nucleotides are 2'-O-methyl adenosine.
[0081] The invention also relates to an aptamer composition
comprising a sequence where substantially all adenosine nucleotides
are 2'-O-methyl adenosine, substantially all cytidine nucleotides
are 2'-O-methyl cytidine, substantially all guanosine nucleotides
are 2'-O-methyl guanosine or deoxy guanosine, substantially all
uridine nucleotides are 2'-O-methyl uridine, where less than about
10% of the guanosine nucleotides are deoxy guanosine. In one
embodiment, the aptamer has a sequence composition where at least
80% of all adenosine nucleotides are 2'-O-methyl adenosine, at
least 80% of all cytidine nucleotides are 2'-O-methyl cytidine, at
least 80% of all guanosine nucleotides are 2'-O-methyl guanosine,
at least 80% of all uridine nucleotides are 2'-O-methyl uridine,
and no more than about 10% of all guanosine nucleotides are deoxy
guanosine. In another embodiment, the aptamer has a sequence
composition where at least 90% of all adenosine nucleotides are
2'-O-methyl adenosine, at least 90% of all cytidine nucleotides are
2'-O-methyl cytidine, at least 90% of all guanosine nucleotides are
2'-O-methyl guanosine, at least 90% of all uridine nucleotides are
2'-O-methyl uridine, and no more than about 10% of all guanosine
nucleotides are deoxy guanosine. In another embodiment, the aptamer
has a sequence composition where 100% of all adenosine nucleotides
are 2'-O-methyl adenosine, 100% of all cytidine nucleotides are
2'-O-methyl cytidine, at least 90% of all guanosine nucleotides are
2'-O-methyl guanosine, 100% of all uridine nucleotides are
2'-O-methyl uridine, and no more than about 10% of all guanosine
nucleotides are deoxy guanosine.
[0082] The invention also relates to an aptamer composition
comprising a sequence where substantially all adenosine nucleotides
are 2'-O-methyl adenosine, substantially all uridine nucleotides
are 2'-O-methyl uridine, substantially all cytidine nucleotides are
2'-O-methyl cytidine, and substantially all guanosine nucleotides
are 2'-F guanosine sequence. In one embodiment, the aptamer has a
sequence composition where at least 80% of all adenosine
nucleotides are 2'-O-methyl adenosine, at least 80% of all uridine
nucleotides are 2'-O-methyl uridine, at least 80% of all cytidine
nucleotides are 2'-O-methyl cytidine, and at least 80% of all
guanosine nucleotides are 2'-F guanosine. In another embodiment,
the aptamer has a sequence composition where at least 90% of all
adenosine nucleotides are 2'-O-methyl adenosine, at least 90% of
all uridine nucleotides are 2'-O-methyl uridine, at least 90% of
all cytidine nucleotides are 2'-O-methyl cytidine, and at least 90%
of all guanosine nucleotides are 2'-F guanosine. In another
embodiment, the aptamer has a sequence composition where 100% of
all adenosine nucleotides are 2'-O-methyl adenosine, 100% of all
uridine nucleotides are 2'-O-methyl uridine, 100% of all cytidine
nucleotides are 2'-O-methyl cytidine, and 100% of all guanosine
nucleotides are 2'-F guanosine.
[0083] The invention also relates to an aptamer composition
comprising a sequence where substantially all adenosine nucleotides
are 2'-deoxy adenosine, substantially all cytidine nucleotides are
2'-O-methyl cytidine, substantially all guanosine nucleotides are
2'-O-methyl guanosine, and substantially all uridine nucleotides
are 2'-O-methyl uridine. In one embodiment, the aptamer has a
sequence composition where at least 80% of all adenosine
nucleotides are 2'-deoxy adenosine, at least 80% of all cytidine
nucleotides are 2'-O-methyl cytidine, at least 80% of all guanosine
nucleotides are 2'-O-methyl guanosine, and at least 80% of all
uridine nucleotides are 2'-O-methyl uridine. In another embodiment,
the aptamer has a sequence composition where at least 90% of all
adenosine nucleotides are 2'-deoxy adenosine, at least 90% of all
cytidine nucleotides are 2'-O-methyl cytidine, at least 90% of all
guanosine nucleotides are 2'-O-methyl guanosine, and at least 90%
of all uridine nucleotides are 2'-O-methyl uridine. In another
embodiment, the aptamer has a sequence composition where 100% of
all adenosine nucleotides are 2'-deoxy adenosine, 100% of all
cytidine nucleotides are 2'-O-methyl cytidine, 100% of all
guanosine nucleotides are 2'-O-methyl guanosine, and 100% of all
uridine nucleotides are 2'-O-methyl uridine.
[0084] The invention also relates to an aptamer composition
comprising a sequence where substantially all adenosine nucleotides
are 2'-OH adenosine, substantially all guanosine nucleotides are
2'-OH guanosine, substantially all cytidine nucleotides are 2'-OH
cytidine, and substantially all uridine nucleotides are 2'-OH
uridine. In one embodiment, the aptamer has a sequence composition
where at least 80% of all adenosine nucleotides are 2'-OH
adenosine, at least 80% of all cytidine nucleotides are 2'-OH
cytidine, at least 80% of all guanosine nucleotides are 2'-OH
guanosine, and at least 80% of all uridine nucleotides are 2'-OH
uridine. In another embodiment, the aptamer has a sequence
composition where at least 90% of all adenosine nucleotides are
2'-OH adenosine, at least 90% of all cytidine nucleotides are 2'-OH
cytidine, at least 90% of all guanosine nucleotides are 2'-OH
guanosine, and at least 90% of all uridine nucleotides are 2'-OH
uridine. In another embodiment, the aptamer has a sequence
composition where 100% of all adenosine nucleotides are 2'-OH
adenosine, 100% of all cytidine nucleotides are 2'-OH cytidine,
100% of all guanosine nucleotides are 2'-OH guanosine, and 100% of
all uridine nucleotides are 2'-OH uridine.
DETAILED DESCRIPTION OF THE INVENTION
[0085] The details of one or more embodiments of the invention are
set forth in the accompanying description below. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
Other features, objects, and advantages of the invention will be
apparent from the description. In the specification, the singular
forms also include the plural unless the context clearly dictates
otherwise. Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
In the case of conflict, the present Specification will
control.
[0086] Modified Nucleotide Transcription
[0087] The present invention provides materials and methods to
produce stabilized oligonucleotides (including, e.g., aptamers)
that contain modified nucleotides (e.g., nucleotides which have a
modification at the 2'position) which make the oligonucleotide more
stable than the unmodified oligonucleotide. The stabilized
oligonucleotides produced by the materials and methods of the
present invention are also more stable to enzymatic and chemical
degradation as well as thermal and physical degradation.
[0088] In order for an aptamer to be suitable for use as a
therapeutic, it is preferably inexpensive to synthesize, safe and
stable in vivo. Wild-type RNA and DNA aptamers are typically not
stable in vivo because of their susceptibility to degradation by
nucleases. Resistance to nuclease degradation can be greatly
increased by the incorporation of modifying groups at the
2'-position. Fluoro and amino groups have been successfully
incorporated into oligonucleotide libraries from which aptamers
have been subsequently selected. However, these modifications
greatly increase the cost of synthesis of the resultant aptamer,
and may introduce safety concerns because of the possibility that
the modified nucleotides could be recycled into host DNA, by
degradation of the modified oligonucleotides and subsequent use of
the nucleotides as substrates for DNA synthesis.
[0089] Aptamers that contain 2'-O-methyl (2'-OMe) nucleotides
overcome many of these drawbacks. Oligonucleotides containing
2'-O-methyl nucleotides are nuclease-resistant and inexpensive to
synthesize. Although 2'-O-methyl nucleotides are ubiquitous in
biological systems, natural polymerases do not accept 2'-O-methyl
NTPs as substrates under physiological conditions, thus there are
no safety concerns over the recycling of 2'-O-methyl nucleotides
into host DNA. A generic formula for a 2'-OMe nucleotide is shown
in FIG. 2.
[0090] There are several examples of 2'-O-Mecontaining aptamers in
the literature, see, for example Green et al., Current Biology 2,
683-695, 1995. These were generated by the in vitro selection of
libraries of modified transcripts in which the C and U residues
were 2'-fluoro (2'-F) substituted and the A and G residues were
2'-OH. Once functional sequences were identified then each A and G
residue was tested for tolerance to 2'-OMe substitution, and the
aptamer was re-synthesized having all A and G residues which
tolerated 2'-OMe substitution as 2'-OMe residues. Most of the A and
G residues of aptamers generated in this two-step fashion tolerate
substitution with 2'-OMe residues, although, on average,
approximately 20% do not. Consequently, aptamers generated using
this method tend to contain from two to four 2'-OH residues, and
stability and cost of synthesis are compromised as a result. By
incorporating modified nucleotides into the transcription reaction
which generate stabilized oligonucleotides used in oligonucleotide
libraries from which aptamers are selected and enriched by
SELEX.TM. (and/or any of its variations and improvements, including
those described below), the methods of the current invention
eliminate the need for stabilizing the selected aptamer
oligonucleotides (e.g., by resynthesizing the aptamer
oligonucleotides with modified nucleotides).
[0091] Furthermore, the modified oligonucleotides of the invention
can be further stabilized after the selection process has been
completed. (See "post-SELEX.TM. modifications", including
truncating, deleting and modification, below.)
[0092] The SELEX.TM. Method
[0093] A suitable method for generating an aptamer is with the
process entitled "Systematic Evolution of Ligands by EXponential
enrichment" ("SELEX.TM.") depicted generally in FIG. 1. The
SELEX.TM. process is a method for the in vitro evolution of nucleic
acid molecules with highly specific binding to target molecules and
is described in, e.g., U.S. patent application Ser. No. 07/536,428,
filed Jun. 11, 1990, now abandoned, U.S. Pat. No. 5,475,096
entitled "Nucleic Acid Ligands", and U.S. Pat. No. 5,270,163 (see
also WO 91/19813) entitled "Nucleic Acid Ligands". Each
SELEX.TM.-identified nucleic acid ligand is a specific ligand of a
given target compound or molecule. The SELEX.TM. process is based
on the unique insight that nucleic acids have sufficient capacity
for forming a variety of two- and three-dimensional structures and
sufficient chemical versatility available within their monomers to
act as ligands (form specific binding pairs) with virtually any
chemical compound, whether monomeric or polymeric. Molecules of any
size or composition can serve as targets.
[0094] SELEX.TM. relies as a starting point upon a large library of
single stranded oligonucleotide templates comprising randomized
sequences derived from chemical synthesis on a standard DNA
synthesizer. In some examples, a population of 100% random
oligonucleotides is screened. In others, each oligonucleotide in
the population comprises a random sequence and at least one fixed
sequence at its 5' and/or 3' end which comprises a sequence shared
by all the molecules of the oligonucleotide population. Fixed
sequences include sequences such as hybridization sites for PCR
primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7,
SP6, and the like), restriction sites, or homopolymeric sequences,
such as poly A or poly T tracts, catalytic cores, sites for
selective binding to affinity columns, and other sequences to
facilitate cloning and/or sequencing of an oligonucleotide of
interest.
[0095] The random sequence portion of the oligonucleotide can be of
any length and can comprise ribonucleotides and/or
deoxyribonucleotides and can include modified or non-natural
nucleotides or nucleotide analogs. See, e.g., U.S. Pat. Nos.
5,958,691; 5,660,985; 5,958,691; 5,698,687; 5,817,635; and
5,672,695, and PCT publication WO 92/07065. Random oligonucleotides
can be synthesized from phosphodiester-linked nucleotides using
solid phase oligonucleotide synthesis techniques well known in the
art (Froehler et al., Nucl. Acid Res. 14:5399-5467 (1986); Froehler
et al., Tet. Lett. 27:5575-5578 (1986)). Oligonucleotides can also
be synthesized using solution phase methods such as triester
synthesis methods (Sood et al., Nucl. Acid Res. 4:2557 (1977);
Hirose et al., Tet. Lett., 28:2449 (1978)). Typical syntheses
carried out on automated DNA synthesis equipment yield
10.sup.15-10.sup.17 molecules. Sufficiently large regions of random
sequence in the sequence design increases the likelihood that each
synthesized molecule is likely to represent a unique sequence.
[0096] To synthesize randomized sequences, mixtures of all four
nucleotides are added at each nucleotide addition step during the
synthesis process, allowing for random incorporation of
nucleotides. In one embodiment, random oligonucleotides comprise
entirely random sequences; however, in other embodiments, random
oligonucleotides can comprise stretches of nonrandom or partially
random sequences. Partially random sequences can be created by
adding the four nucleotides in different molar ratios at each
addition step.
[0097] Template molecules typically contain fixed 5' and 3'
terminal sequences which flank an internal region of 30-50 random
nucleotides. A standard (1 .mu.mole) scale synthesis will yield
10.sup.15-10.sup.16 individual template molecules, sufficient for
most SELEX.TM. experiments. The RNA library is generated from this
starting library by in vitro transcription using recombinant T7 RNA
polymerase. This library is then mixed with the target under
conditions favorable for binding and subjected to step-wise
iterations of binding, partitioning and amplification, using the
same general selection scheme, to achieve virtually any desired
criterion of binding affinity and selectivity. Starting from a
mixture of nucleic acids, preferably comprising a segment of
randomized sequence, the SELEX.TM. method includes steps of
contacting the mixture with the target under conditions favorable
for binding, partitioning unbound nucleic acids from those nucleic
acids which have bound specifically to target molecules,
dissociating the nucleic acid-target complexes, amplifying the
nucleic acids dissociated from the nucleic acid-target complexes to
yield a ligand-enriched mixture of nucleic acids, then reiterating
the steps of binding, partitioning, dissociating and amplifying
through as many cycles as desired to yield highly specific high
affinity nucleic acid ligands to the target molecule.
[0098] Within a nucleic acid mixture containing a large number of
possible sequences and structures, there is a wide range of binding
affinities for a given target. A nucleic acid mixture comprising,
for example a 20 nucleotide randomized segment containing only
natural unmodified nucleotides can have 420 candidate
possibilities. Those which have the higher affinity constants for
the target are most likely to bind to the target. After
partitioning, dissociation and amplification, a second nucleic acid
mixture is generated, enriched for the higher binding affinity
candidates. Additional rounds of selection progressively favor the
best ligands until the resulting nucleic acid mixture is
predominantly composed of only one or a few sequences. These can
then be cloned, sequenced and individually tested for binding
affinity as pure ligands.
[0099] Cycles of selection and amplification are repeated until a
desired goal is achieved. In the most general case,
selection/amplification is continued until no significant
improvement in binding strength is achieved on repetition of the
cycle. The method may be used to sample as many as about 10.sup.18
different nucleic acid species. The nucleic acids of the test
mixture preferably include a randomized sequence portion as well as
conserved sequences necessary for efficient amplification. Nucleic
acid sequence variants can be produced in a number of ways
including synthesis of randomized nucleic acid sequences and size
selection from randomly cleaved cellular nucleic acids. The
variable sequence portion may contain fully or partially random
sequence; it may also contain subportions of conserved sequence
incorporated with randomized sequence. Sequence variation in test
nucleic acids can be introduced or increased by mutagenesis before
or during the selection/amplification iterations.
[0100] In one embodiment of SELEX.TM., the selection process is so
efficient at isolating those nucleic acid ligands that bind most
strongly to the selected target, that only one cycle of selection
and amplification is required. Such an efficient selection may
occur, for example, in a chromatographic-type process wherein the
ability of nucleic acids to associate with targets bound on a
column operates in such a manner that the column is sufficiently
able to allow separation and isolation of the highest affinity
nucleic acid ligands.
[0101] In many cases, it is not necessarily desirable to perform
the iterative steps of SELEX.TM. until a single nucleic acid ligand
is identified. The target-specific nucleic acid ligand solution may
include a family of nucleic acid structures or motifs that have a
number of conserved sequences and a number of sequences which can
be substituted or added without significantly affecting the
affinity of the nucleic acid ligands to the target. By terminating
the SELEX.TM. process prior to completion, it is possible to
determine the sequence of a number of members of the nucleic acid
ligand solution family.
[0102] A variety of nucleic acid primary, secondary and tertiary
structures are known to exist. The structures or motifs that have
been shown most commonly to be involved in non-Watson-Crick type
interactions are referred to as hairpin loops, symmetric and
asymmetric bulges, pseudoknots and myriad combinations of the same.
Almost all known cases of such motifs suggest that they can be
formed in a nucleic acid sequence of no more than 30 nucleotides.
For this reason, it is often preferred that SELEX.TM. procedures
with contiguous randomized segments be initiated with nucleic acid
sequences containing a randomized segment of between about 20-50
nucleotides.
[0103] The core SELEX.TM. method has been modified to achieve a
number of specific objectives. For example, U.S. Pat. No. 5,707,796
describes the use of SELEX.TM. in conjunction with gel
electrophoresis to select nucleic acid molecules with specific
structural characteristics, such as bent DNA. U.S. Pat. No.
5,763,177 describes SELEX.TM. based methods for selecting nucleic
acid ligands containing photoreactive groups capable of binding
and/or photocrosslinking to and/or photoinactivating a target
molecule. U.S. Pat. No. 5,567,588 and U.S. application Ser. No.
08/792,075, filed Jan. 31, 1997, entitled "Flow Cell SELEX.TM.",
describe SELEX.TM. based methods which achieve highly efficient
partitioning between oligonucleotides having high and low affinity
for a target molecule. U.S. Pat. No. 5,496,938 describes methods
for obtaining improved nucleic acid ligands after the SELEX.TM.
process has been performed. U.S. Pat. No. 5,705,337 describes
methods for covalently linking a ligand to its target.
[0104] SELEX.TM. can also be used to obtain nucleic acid ligands
that bind to more than one site on the target molecule, and to
obtain nucleic acid ligands that include non-nucleic acid species
that bind to specific sites on the target. SELEX.TM. provides means
for isolating and identifying nucleic acid ligands which bind to
any envisionable target, including large and small biomolecules
including proteins (including both nucleic acid-binding proteins
and proteins not known to bind nucleic acids as part of their
biological function) cofactors and other small molecules. For
example, see U.S. Pat. No. 5,580,737 which discloses nucleic acid
sequences identified through SELEX.TM. which are capable of binding
with high affinity to caffeine and the closely related analog,
theophylline.
[0105] Counter-SELEX.TM. is a method for improving the specificity
of nucleic acid ligands to a target molecule by eliminating nucleic
acid ligand sequences with cross-reactivity to one or more
non-target molecules. Counter-SELEX.TM. is comprised of the steps
of a) preparing a candidate mixture of nucleic acids; b) contacting
the candidate mixture with the target, wherein nucleic acids having
an increased affinity to the target relative to the candidate
mixture may be partitioned from the remainder of the candidate
mixture; c) partitioning the increased affinity nucleic acids from
the remainder of the candidate mixture; d) contacting the increased
affinity nucleic acids with one or more non-target molecules such
that nucleic acid ligands with specific affinity for the non-target
molecule(s) are removed; and e) amplifying the nucleic acids with
specific affinity to the target molecule to yield a mixture of
nucleic acids enriched for nucleic acid sequences with a relatively
higher affinity and specificity for binding to the target
molecule.
[0106] One potential problem encountered in the use of nucleic
acids as therapeutics and vaccines is that oligonucleotides in
their phosphodiester form may be quickly degraded in body fluids by
intracellular and/or extracellular enzymes such as endonucleases
and exonucleases before the desired effect is manifest. SELEX.TM.
methods therefore encompass the identification of high-affinity
nucleic acid ligands which are altered, after selection, to contain
modified nucleotides which confer improved characteristics on the
ligand, such as improved in vivo stability or improved delivery
characteristics. Modifications of nucleic acid ligands include, but
are not limited to, those which provide other chemical groups that
incorporate additional charge, polarizability, hydrophobicity,
hydrogen bonding, electrostatic interaction, and fluxionality to
the nucleic acid ligand bases or to the nucleic acid ligand as a
whole. Modifications include chemical substitutions at the ribose
and/or phosphate and/or base positions, such as 2'-position sugar
modifications, 5-position pyrimidine modifications, 8-position
purine modifications, modifications at exocyclic amines,
substitution of 4-thiouridine, substitution of 5-bromo or
5-iodo-uracil; backbone modifications, phosphorothioate or alkyl
phosphate modifications, methylations, unusual base-pairing
combinations such as the isobases isocytidine and isoguanidine and
the like. Modifications can also include 3' and 5' modifications
such as capping.
[0107] In oligonucleotides which comprise modified sugar groups,
for example, one or more of the hydroxyl groups is replaced with
halogen, aliphatic groups, or functionalized as ethers or amines.
Examples of substitution at the 2'-posititution of the furanose
residue include O-alkyl (e.g., O-methyl), O-allyl, S-alkyl,
S-allyl, or a halo group. Methods of synthesis of 2'-modified
sugars are described in Sproat, et al., Nucl. Acid Res. 19:733-738
(1991); Cotten, et al., Nucl. Acid Res. 19:2629-2635 (1991); and
Hobbs, et al., Biochemistry 12:5138-5145 (1973). Other
modifications are known to one of ordinary skill in the art.
[0108] SELEX.TM.-identified nucleic acid ligands synthesized after
selection to contain modified nucleotides are described in U.S.
Pat. No. 5,660,985, which describes oligonucleotides containing
nucleotide derivatives chemically modified at the 5' and 2'
positions of pyrimidines. Additionally, U.S. Pat. No. 5,756,703
describes oligonucleotides containing various 2'-modified
pyrimidines; and U.S. Pat. No. 5,580,737 describes highly specific
nucleic acid ligands containing one or more nucleotides modified
with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl
(2'-OMe) substituents.
[0109] The SELEX.TM. method encompasses combining selected
oligonucleotides with other selected oligonucleotides and
non-oligonucleotide functional units as described in U.S. Pat. No.
5,637,459 and U.S. Pat. No. 5,683,867. The SELEX.TM. method further
encompasses combining selected nucleic acid ligands with lipophilic
or non-immunogenic high molecular weight compounds in a diagnostic
or therapeutic complex, as described in U.S. Pat. No. 6,011,020.
VEGF nucleic acid ligands that are associated with a lipophilic
compound, such as diacyl glycerol or dialkyl glycerol, in a
diagnostic or therapeutic complex are described in U.S. Pat. No.
5,859,228.
[0110] VEGF nucleic acid ligands that are associated with a
lipophilic compound, such as a glycerol lipid, or a non-immunogenic
high molecular weight compound, such as polyalkylene glycol are
further described in U.S. Pat. No. 6,051,698. VEGF nucleic acid
ligands that are associated with a non-immunogenic, high molecular
weight compound or a lipophilic compound are further described in
PCT Publication No. WO 98/18480. These patents and applications
describe the combination of a broad array of oligonucleotide shapes
and other properties, and the efficient amplification and
replication properties, of oligonucleotides with the desirable
properties of other molecules.
[0111] The identification of nucleic acid ligands to small,
flexible peptides via the SELEX.TM. method has also been explored.
Small peptides have flexible structures and usually exist in
solution in an equilibrium of multiple conformers, and thus it was
initially thought that binding affinities may be limited by the
conformational entropy lost upon binding a flexible peptide.
However, the feasibility of identifying nucleic acid ligands to
small peptides in solution was demonstrated in U.S. Pat. No.
5,648,214. In this patent, high affinity RNA nucleic acid ligands
to substance P, an 11 amino acid peptide, were identified.
[0112] To generate oligonucleotide populations which are resistant
to nucleases and hydrolysis, modified oligonucleotides can be used
and can include one or more substitute internucleotide linkages,
altered sugars, altered bases, or combinations thereof. In one
embodiment, oligonucleotides are provided in which the P(O)O group
is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), P(O)NR.sub.2
("amidate"), P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal") or
3'-amine (--NH--CH.sub.2--CH.sub.2--), wherein each R or R' is
independently H or substituted or unsubstituted alkyl. Linkage
groups can be attached to adjacent nucleotide through an --O--,
--N--, or --S-- linkage. Not all linkages in the oligonucleotide
are required to be identical.
[0113] Nucleic acid aptamer molecules are generally selected in a 5
to 20 cycle procedure. In one embodiment, heterogeneity is
introduced only in the initial selection stages and does not occur
throughout the replicating process.
[0114] The starting library of DNA sequences is generated by
automated chemical synthesis on a DNA synthesizer. This library of
sequences is transcribed in vitro into RNA using T7 RNA polymerase
or a modified T7 RNA polymerase, and purified. In one example, the
5'-fixed:random:3'-fixe- d sequence includes a random sequence
having from 30 to 50 nucleotides.
[0115] Incorporation of modified nucleotides into the aptamers of
the invention is accomplished before (pre-) the selection process
(e.g., a pre-SELEX.TM. process modification). Optionally, aptamers
of the invention in which modified nucleotides have been
incorporated by pre-SELEX.TM. process modification can be further
modified by post-SELEX.TM. process modification (i.e., a
post-SELEX.TM. process modification after a pre-SELEX.TM.
modification). Pre-SELEX.TM. process modifications yield modified
nucleic acid ligands with specificity for the SELEX.TM. target and
also improved in vivo stability. Post-SELEX.TM. process
modifications (e.g., modification of previously identified ligands
having nucleotides incorporated by pre-SELEX.TM. process
modification) can result in a further improvement of in vivo
stability without adversely affecting the binding capacity of the
nucleic acid ligand having nucleotides incorporated by
pre-SELEX.TM. process modification.
[0116] Modified Polymerases
[0117] A single mutant T7 polymerase (Y639F) in which the tyrosine
residue at position 639 has been changed to phenylalanine readily
utilizes 2'deoxy, 2'amino-, and 2'fluoro-nucleotide triphosphates
(NTPs) as substrates and has been widely used to synthesize
modified RNAs for a variety of applications. However, this mutant
T7 polymerase reportedly can not readily utilize (e.g.,
incorporate) NTPs with bulkier 2'-substituents, such as 2'-O-methyl
(2'-OMe) or 2'-azido (2'-N.sub.3) substituents. For incorporation
of bulky 2' substituents, a double T7 polymerase mutant
(Y639F/H784A) having the histidine at position 784 changed to an
alanine, or other small amino acid, residue, in addition to the
Y639F mutation has been described and has been used to incorporate
modified pyrimidine NTPs. A single mutant T7 polymerase (H784A)
having the histidine at position 784 changed to an alanine residue
has also been described. (Padilla et al., Nucleic Acids Research,
2002, 30: 138). In both the Y639F/H784A double mutant and H784A
single mutant T7 polymerases, the change to smaller amino acid
residues allows for the incorporation of bulkier nucleotide
substrates, e.g., 2'-O methyl substituted nucleotides.
[0118] The present invention provides methods and conditions for
using these and other modified T7 polymerases having a higher
incorporation rate of modified nucleotides having bulky
substituents at the furanose 2' position, than wild-type
polymerases. Generally, it has been found that under the conditions
disclosed herein, the Y693F single mutant can be used for the
incorporation of all 2'-OMe substituted NTPs except GTP and the
Y639F/H784A double mutant can be used for the incorporation of all
2'-OMe substituted NTPs including GTP. It is expected that the
H784A single mutant possesses similar properties when used under
the conditions disclosed herein.
[0119] The present invention provides methods and conditions for
modified T7 polymerases to enzymatically incorporate modified
nucleotides into oligonucleotides. Such oligonucleotides may be
synthesized entirely of modified nucleotides, or with a subset of
modified nucleotides. The modifications can be the same or
different. All nucleotides may be modified, and all may contain the
same modification. All nucleotides may be modified, but contain
different modifications, e.g., all nucleotides containing the same
base may have one type of modification, while nucleotides
containing other bases may have different types of modification.
All purine nucleotides may have one type of modification (or are
unmodified), while all pyrimidine nucleotides have another,
different type of modification (or are unmodified). In this way,
transcripts, or libraries of transcripts are generated using any
combination of modifications, for example, ribonucleotides, (2'-OH,
"rN"), deoxyribonucleotides (2'-deoxy), 2'-F, and 2'-OMe
nucleotides. A mixture containing 2'-OMe C and U and 2'-OH A and G
is called "rRmY"; a mixture containing deoxy A and G and 2'-OMe U
and C is called "dRmY"; a mixture containing 2'-OMe A, C, and U,
and 2'-OH G is called "rGmH"; a mixture alternately containing
2'-OMe A, C, U and G and 2'-OMe A, U and C and 2'-F G is called
"toggle"; a mixture containing 2'-OMe A, U, C, and G, where up to
10% of the G's are deoxy is called "r/mGmH"; a mixture containing
2'-O Me A, U, and C, and 2'-F G is called "fGmH"; and a mixture
containing deoxy A, and 2'-OMe C, G and U is called "dAmB".
[0120] A preferred embodiment includes any combination of 2'-OH,
2'-deoxy and 2'-OMe nucleotides. A more preferred embodiment
includes any combination of 2'-deoxy and 2'-OMe nucleotides. An
even more preferred embodiment is with any combination of 2'-deoxy
and 2'-OMe nucleotides in which the pyrimidines are 2'-OMe (such as
dRmY, mN or dGmH).
[0121] 2'-Modified SELEX.TM.
[0122] The present invention provides methods to generate libraries
of 2'-modified (e.g., 2'-OMe) RNA transcripts in conditions under
which a polymerase accepts 2'-modified NTPs. Preferably, the
polymerase is the Y693F/H784A double mutant or the Y693F single
mutant. Other polymerases, particularly those that exhibit a high
tolerance for bulky 2'-substituents, may also be used in the
present invention. Such polymerases can be screened for this
capability by assaying their ability to incorporate modified
nucleotides under the transcription conditions disclosed herein. A
number of factors have been determined to be crucial for the
transcription conditions useful in the methods disclosed herein.
For example, great increases in the yields of modified transcript
are observed when a leader sequence is incorporated into the 5' end
of a fixed sequence at the 5' end of the DNA transcription
template, such that at least about the first 6 residues of the
resultant transcript are all purines.
[0123] Another important factor in obtaining transcripts
incorporating modified nucleotides is the presence or concentration
of 2'-OH GTP. Transcription can be divided into two phases: the
first phase is initiation, during which an NTP is added to the
3'-hydroxyl end of GTP (or another substituted guanosine) to yield
a dinucleotide which is then extended by about 10-12 nucleotides,
the second phase is elongation, during which transcription proceeds
beyond the addition of the first about 10-12 nucleotides. It has
been found that small amounts of 2'-OH GTP added to a transcription
mixture containing an excess of 2'-OMe GTP are sufficient to enable
the polymerase to initiate transcription using 2'-OH GTP, but once
transcription enters the elongation phase the reduced
discrimination between 2'-OMe and 2'-OH GTP, and the excess of
2'-OMe GTP over 2'-OH GTP allows the incorporation of principally
the 2'-OMe GTP.
[0124] Another important factor in the incorporation of 2'-OMe into
transcripts is the use of both divalent magnesium and manganese in
the transcription mixture. Different combinations of concentrations
of magnesium chloride and manganese chloride have been found to
affect yields of 2'-O-methylated transcripts, the optimum
concentration of the magnesium and manganese chloride being
dependent on the concentration in the transcription reaction
mixture of NTPs which complex divalent metal ions. To obtain the
greatest yields of maximally 2' substituted O-methylated
transcripts (i.e., all A, C, and U and about 90% of G nucleotides),
concentrations of approximately 5 mM magnesium chloride and 1.5 mM
manganese chloride are preferred when each NTP is present at a
concentration of 0.5 mM. When the concentration of each NTP is 1.0
mM, concentrations of approximately 6.5 mM magnesium chloride and
2.0 mM manganese chloride are preferred. When the concentration of
each NTP is 2.0 mM, concentrations of approximately 9.6 mM
magnesium chloride and 2.9 mM manganese chloride are preferred. In
any case, departures from these concentrations of up to two-fold
still give significant amounts of modified transcripts.
[0125] Priming transcription with GMP or guanosine is also
important. This effect results from the specificity of the
polymerase for the initiating nucleotide. As a result, the
5'-terminal nucleotide of any transcript generated in this fashion
is likely to be 2'-OH G. The preferred concentration of GMP (or
guanosine) is 0.5 mM and even more preferably 1 mM. It has also
been found that including PEG, preferably PEG-8000, in the
transcription reaction is useful to maximize incorporation of
modified nucleotides.
[0126] For maximum incorporation of 2'-OMe ATP (100%), UTP(100%),
CTP(100%) and GTP (.about.90%) ("r/mGmH") into transcripts the
following conditions are preferred: HEPES buffer 200 mM, DTT 40 mM,
spermidine 2 mM, PEG-8000 10% (w/v), Triton X-100 0.01% (w/v),
MgCl.sub.2 5 mM (6.5 mM where the concentration of each 2'-OMe NTP
is 1.0 mM), MnCl.sub.2 1.5 mM (2.0 mM where the concentration of
each 2'-OMe NTP is 1.0 mM), 2'-OMe NTP (each) 500 .mu.M (more
preferably, 1.0 mM), 2'-OH GTP 30 .mu.M, 2'-OH GMP 500 .mu.M, pH
7.5, Y639F/H784A T7 RNA Polymerase 15 units/ml, inorganic
pyrophosphatase 5 units/ml, and an all-purine leader sequence of at
least 8 nucleotides long. As used herein, one unit of the
Y639F/H784A mutant T7 RNA polymerase, or any other mutant T7 RNA
polymerase specified herein) is defined as the amount of enzyme
required to incorporate 1 nmole of 2'-OMe NTPs into transcripts
under the r/mGmH conditions. As used herein, one unit of inorganic
pyrophosphatase is defined as the amount of enzyme that will
liberate 1.0 mole of inorganic orthophosphate per minute at pH 7.2
and 25.degree. C.
[0127] For maximum incorporation (100%) of 2'-OMe ATP, UTP and CTP
("rGmH") into transcripts the following conditions are preferred:
HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10%
(w/v), Triton X-100 0.01% (w/v), MgCl.sub.2 5 mM (9.6 mM where the
concentration of each 2'-OMe NTP is 2.0 mM), MnCl.sub.2 1.5 mM (2.9
mM where the concentration of each 2'-OMe NTP is 2.0 mM), 2'-OMe
NTP (each) 500 .mu.M (more preferably, 2.0 mM), pH 7.5, Y639F T7
RNA Polymerase 15 units/ml, inorganic pyrophosphatase 5 units/ml,
and an all-purine leader sequence of at least 8 nucleotides
long.
[0128] For maximum incorporation (100%) of 2'-OMe UTP and CTP
("rRmY") into transcripts the following conditions are preferred:
HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10%
(w/v), Triton X-100 0.01% (w/v), MgCl.sub.2 5 mM (9.6 mM where the
concentration of each 2'-OMe NTP is 2.0 mM), MnCl.sub.2 1.5 mM (2.9
mM where the concentration of each 2'-OMe NTP is 2.0 mM), 2'-OMe
NTP (each) 500 .mu.M (more preferably, 2.0 mM), pH 7.5, Y639F/H784A
T7 RNA Polymerase 15 units/ml, inorganic pyrophosphatase 5
units/ml, and an all-purine leader sequence of at least 8
nucleotides long.
[0129] For maximum incorporation (100%) of deoxy ATP and GTP and
2'-OMe UTP and CTP ("dRmY") into transcripts the following
conditions are preferred: HEPES buffer 200 mM, DTT 40 mM,
spermidine 2 mM, PEG-8000 10% (w/v), Triton X-100 0.01% (w/v),
MgCl.sub.2 9.6 mM, MnCl.sub.2 2.9 mM, 2'-OMe NTP (each) 2.0 mM, pH
7.5, Y639F T7 RNA Polymerase 15 units/ml, inorganic pyrophosphatase
5 units/ml, and an all-purine leader sequence of at least 8
nucleotides long.
[0130] For maximum incorporation (100%) of 2'-OMe ATP, UTP and CTP
and 2'-F GTP ("fGmH") into transcripts the following conditions are
preferred: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM,
PEG-8000 10% (w/v), Triton X-100 0.01% (w/v), MgCl.sub.2 9.6 mM,
MnCl.sub.2 2.9 mM, 2'-OMe NTP (each) 2.0 mM, pH 7.5, Y639F T7 RNA
Polymerase 15 units/ml, inorganic pyrophosphatase 5 units/ml, and
an all-purine leader sequence of at least 8 nucleotides long.
[0131] For maximum incorporation (100%) of deoxy ATP and 2'-OMe
UTP, GTP and CTP ("dAmB") into transcripts the following conditions
are preferred: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM,
PEG-8000 10% (w/v), Triton X-100 0.01% (w/v), MgCl.sub.2 9.6 mM,
MnCl.sub.2 2.9 mM, 2'-OMe NTP (each) 2.0 mM, pH 7.5, Y639F T7 RNA
Polymerase 15 units/ml, inorganic pyrophosphatase 5 units/ml, and
an all-purine leader sequence of at least 8 nucleotides long.
[0132] For each of the above, (1) transcription is preferably
performed at a temperature of from about 30.degree. C. to about
45.degree. C. and for a period of at least two hours and (2) 50-300
nM of a double stranded DNA transcription template is used (200 nm
template was used for round 1 to increase diversity (300 nm
template was used for dRmY transcriptions), and for subsequent
rounds approximately 50 nM, a {fraction (1/10)} dilution of an
optimized PCR reaction, using conditions described herein, was
used). The preferred DNA transcription templates are described
below (where ARC254 and ARC256 transcribe under all 2'-OMe
conditions and ARC255 transcribes under rRmY conditions).
ARC254:
1 ARC254: 5'-CATCGATGCTAGTCGTAACGATCCNNNNNNN (SEQ ID NO:1)
NNNNNNNNNNNNNNNNNNNNNNNCGAGAACGTTC TCTCCTCTCCCTATAGTGAGTCGTATTA-3'
ARC255: 5'-CATGCATCGCGACTGACTAGCCGNNNNNNNN (SEQ ID NO:2)
NNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCT CTCCTCTCCCTATAGTGAGTCGTATTA-3'
ARC256: 5'-CATCGATCGATCGATCGACAGCGNNNNNNNN (SEQ ID NO:453)
NNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCT
CTCCTCTCCCTATAGTGAGTCGTATTA-3'
[0133] Under rN transcription conditions of the present invention,
the transcription reaction mixture comprises 2'-OH adenosine
triphosphates (ATP), 2'-OH guanosine triphosphates (GTP), 2'-OH
cytidine triphosphates (CTP), and 2'-OH uridine triphosphates
(UTP). The modified oligonucleotides produced using the rN
transcription mixtures of the present invention comprise
substantially all 2'-OH adenosine, 2'-OH guanosine, 2'-OH cytidine,
and 2'-OH uridine. In a preferred embodiment of rN transcription,
the resulting modified oligonucleotides comprise a sequence where
at least 80% of all adenosine nucleotides are 2'-OH adenosine, at
least 80% of all guanosine nucleotides are 2'-OH guanosine, at
least 80% of all cytidine nucleotides are 2'-OH cytidine, and at
least 80% of all uridine nucleotides are 2'-OH uridine. In a more
preferred embodiment of rN transcription, the resulting modified
oligonucleotides of the present invention comprise a sequence where
at least 90% of all adenosine nucleotides are 2'-OH adenosine, at
least 90% of all guanosine nucleotides are 2'-OH guanosine, at
least 90% of all cytidine nucleotides are 2'-OH cytidine, and at
least 90% of all uridine nucleotides are 2'-OH uridine. In a most
preferred embodiment of rN transcription, the modified
oligonucleotides of the present invention comprise 100% of all
adenosine nucleotides are 2'-OH adenosine, of all guanosine
nucleotides are 2'-OH guanosine, of all cytidine nucleotides are
2'-OH cytidine, and of all uridine nucleotides are 2'-OH
uridine.
[0134] Under rRmY transcription conditions of the present
invention, the transcription reaction mixture comprises 2'-OH
adenosine triphosphates, 2'-OH guanosine triphosphates, 2'-O-methyl
cytidine triphosphates, and 2'-O-methyl uridine triphosphates. The
modified oligonucleotides produced using the rRmY transcription
mixtures of the present invention comprise substantially all 2'-OH
adenosine, 2'-OH guanosine, 2'-O-methyl cytidine and 2'-O-methyl
uridine. In a preferred embodiment, the resulting modified
oligonucleotides comprise a sequence where at least 80% of all
adenosine nucleotides are 2'-OH adenosine, at least 80% of all
guanosine nucleotides are 2'-OH guanosine, at least 80% of all
cytidine nucleotides are 2'-O-methyl cytidine and at least 80% of
all uridine nucleotides are 2'-O-methyl uridine. In a more
preferred embodiment, the resulting modified oligonucleotides
comprise a sequence where at least 90% of all adenosine nucleotides
are 2'-OH adenosine, at least 90% of all guanosine nucleotides are
2'-OH guanosine, at least 90% of all cytidine nucleotides are
2'-O-methyl cytidine and at least 90% of all uridine nucleotides
are 2'-O-methyl uridine In a most preferred embodiment, the
resulting modified oligonucleotides comprise a sequence where 100%
of all adenosine nucleotides are 2'-OH adenosine, 100% of all
guanosine nucleotides are 2'-OH guanosine, 100% of all cytidine
nucleotides are 2'-O-methyl cytidine and 100% of all uridine
nucleotides are 2'-O-methyl uridine.
[0135] Under dRmY transcription conditions of the present
invention, the transcription reaction mixture comprises 2'-deoxy
purine triphosphates and 2'-O-methylpyrimidine triphosphates. The
modified oligonucleotides produced using the dRmY transcription
conditions of the present invention comprise substantially all
2'-deoxy purines and 2'-O-methyl pyrimidines. In a preferred
embodiment, the resulting modified oligonucleotides of the present
invention comprise a sequence where at least 80% of all purine
nucleotides are 2'-deoxy purines and at least 80% of all pyrimidine
nucleotides are 2'-O-methylpyrimidines. In a more preferred
embodiment, the resulting modified oligonucleotides of the present
invention comprise a sequence where at least 90% of all purine
nucleotides are 2'-deoxy purines and at least 90% of all pyrimidine
nucleotides are 2'-O-methylpyrimidines. In a most preferred
embodiment, the resulting modified oligonucleotides of the present
invention comprise a sequence where 100% of all purine nucleotides
are 2'-deoxy purines and 100% of all pyrimidine nucleotides are
2'-methylpyrimidines.
[0136] Under rGmH transcription conditions of the present
invention, the transcription reaction mixture comprises 2'-OH
guanosine triphosphates, 2'-O-methyl cytidine triphosphates,
2'-O-methyl uridine triphosphates, and 2'-O-methyl adenosine
triphosphates. The modified oligonucleotides produced using the
rGmH transcription mixtures of the present invention comprise
substantially all 2'-OH guanosine, 2'-O-methyl cytidine,
2'-O-methyl uridine, and 2'-O-methyl adenosine. In a preferred
embodiment, the resulting modified oligonucleotides comprise a
sequence where at least 80% of all guanosine nucleotides are 2'-OH
guanosine, at least 80% of all cytidine nucleotides are 2'-O-methyl
cytidine, at least 80% of all uridine nucleotides are 2'-O-methyl
uridine, and at least 80% of all adenosine nucleotides are
2'-O-methyl adenosine. In a more preferred embodiment, the
resulting modified oligonucleotides comprise a sequence where at
least 90% of all guanosine nucleotides are 2'-OH guanosine, at
least 90% of all cytidine nucleotides are 2'-O-methyl cytidine, at
least 90% of all uridine nucleotides are 2'-O-methyl uridine, and
at least 90% of all adenosine nucleotides are 2'-O-methyl
adenosine. In a most preferred embodiment, the resulting modified
oligonucleotides comprise a sequence where 100% of all guanosine
nucleotides are 2'-OH guanosine, 100% of all cytidine nucleotides
are 2'-O-methyl cytidine, 100% of all uridine nucleotides are
2'-O-methyl uridine, and 100% of all adenosine nucleotides are
2'-O-methyl adenosine.
[0137] Under r/mGmH transcription conditions of the present
invention, the transcription reaction mixture comprises 2'-O-methyl
adenosine triphosphate, 2'-O-methyl cytidine triphosphate,
2'-O-methyl guanosine triphosphate, 2'-O-methyl uridine
triphosphate and deoxy guanosine triphosphate. The resulting
modified oligonucleotides produced using the r/mGmH transcription
mixtures of the present invention comprise substantially all
2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine,
and 2'-O-methyl uridine, wherein the population of guanosine
nucleotides has a maximum of about 10% deoxy guanosine. In a
preferred embodiment, the resulting r/mGmH modified
oligonucleotides of the present invention comprise a sequence where
at least 80% of all adenosine nucleotides are 2'-O-methyl
adenosine, at least 80% of all cytidine nucleotides are 2'-O-methyl
cytidine, at least 80% of all guanosine nucleotides are 2'-O-methyl
guanosine, at least 80% of all uridine nucleotides are 2'-O-methyl
uridine, and no more than about 10% of all guanosine nucleotides
are deoxy guanosine. In a more preferred embodiment, the resulting
modified oligonucleotides comprise a sequence where at least 90% of
all adenosine nucleotides are 2'-O-methyl adenosine, at least 90%
of all cytidine nucleotides are 2'-O-methyl cytidine, at least 90%
of all guanosine nucleotides are 2'-O-methyl guanosine, at least
90% of all uridine nucleotides are 2'-O-methyl uridine, and no more
than about 10% of all guanosine nucleotides are deoxy guanosine. In
a most preferred embodiment, the resulting modified
oligonucleotides comprise a sequence where 100% of all adenosine
nucleotides are 2'-O-methyl adenosine, 100% of all cytidine
nucleotides are 2'-O-methyl cytidine, 90% of all guanosine
nucleotides are 2'-O-methyl guanosine, and 100% of all uridine
nucleotides are 2'-O-methyl uridine, and no more than about 10% of
all guanosine nucleotides are deoxy guanosine.
[0138] Under fGmH transcription conditions of the present
invention, the transcription reaction mixture comprises 2'-O-methyl
adenosine triphosphates (ATP), 2'-O-methyl uridine triphosphates
(UTP), 2'-O-methyl cytidine triphosphates (CTP), and 2'-F guanosine
triphosphates. The modified oligonucleotides produced using the
fGmH transcription conditions of the present invention comprise
substantially all 2'-O-methyl adenosine, 2'-O-methyl uridine,
2'-O-methyl cytidine, and 2'-F guanosine. In a preferred
embodiment, the resulting modified oligonucleotides comprise a
sequence where at least 80% of all adenosine nucleotides are
2-O-methyl adenosine, at least 80% of all uridine nucleotides are
2'-O-methyl uridine, at least 80% of all cytidine nucleotides are
2'-O-methyl cytidine, and at least 80% of all guanosine nucleotides
are 2'-F guanosine. In a more preferred embodiment, the resulting
modified oligonucleotides comprise a sequence where at least 90% of
all adenosine nucleotides are 2'-O-methyl adenosine, at least 90%
of all uridine nucleotides are 2'-O-methyl uridine, at least 90% of
all cytidine nucleotides are 2'-O-methyl cytidine, and at least 90%
of all guanosine nucleotides are 2'-F guanosine. The resulting
modified oligonucleotides comprise a sequence where 100% of all
adenosine nucleotides are 2'-O-methyl adenosine, 100% of all
uridine nucleotides are 2'-O-methyl uridine, 100% of all cytidine
nucleotides are 2'-O-methyl cytidine, and 100% of all guanosine
nucleotides are 2'-F guanosine.
[0139] Under dAmB transcription conditions of the present
invention, the transcription reaction mixture comprises 2'-deoxy
adenosine triphosphates (dATP), 2'-O-methyl cytidine triphosphates
(CTP), 2'-O-methyl guanosine triphosphates (GTP), and 2'-O-methyl
uridine triphosphates (UTP). The modified oligonucleotides produced
using the dAmB transcription mixtures of the present invention
comprise substantially all 2'-deoxy adenosine, 2'-O-methyl
cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine. In a
preferred embodiment, the resulting modified oligonucleotides
comprise a sequence where at least 80% of all adenosine nucleotides
are 2'-deoxy adenosine, at least 80% of all cytidine nucleotides
are 2'-O-methyl cytidine, at least 80% of all guanosine nucleotides
are 2'-O-methyl guanosine, and at least 80% of all uridine
nucleotides are 2'-O-methyl uridine. In a more preferred
embodiment, the resulting modified oligonucleotides comprise a
sequence where at least 90% of all adenosine nucleotides are
2'-deoxy adenosine, at least 90% of all cytidine nucleotides are
2'-O-methyl cytidine, at least 90% of all guanosine nucleotides are
2'-O-methyl guanosine, and at least 90% of all uridine nucleotides
are 2'-O-methyl uridine. In a most preferred embodiment, the
resulting modified oligonucleotides of the present invention
comprise a sequence where 100% of all adenosine nucleotides are
2'-deoxy adenosine, 100% of all cytidine nucleotides are
2'-O-methyl cytidine, 100% of all guanosine nucleotides are
2'-O-methyl guanosine, and 100% of all uridine nucleotides are
2'-O-methyl uridine.
[0140] In each case, the transcription products can then be used as
the library in the SELEX.TM. process to identify aptamers and/or to
determine a conserved motif of sequences that have binding
specificity to a given target. The resulting sequences are already
stabilized, eliminating this step from the process to arrive at a
stabilized aptamer sequence and giving a more highly stabilized
aptamer as a result. Another advantage of the 2'-OMe SELEX.TM.
process is that the resulting sequences are likely to have fewer
2'-OH nucleotides required in the sequence, possibly none.
[0141] As described below, lower but still useful yields of
transcripts fully incorporating 2'-OMe substituted nucleotides can
be obtained under conditions other than the optimized conditions
described above. For example, variations to the above transcription
conditions include:
[0142] The HEPES buffer concentration can range from 0 to 1 M. The
present invention also contemplates the use of other buffering
agents having a pKa between 5 and 10, for example without
limitation, Tris(hydroxymethyl)aminomethane.
[0143] The DTT concentration can range from 0 to 400 mM. The
methods of the present invention also provide for the use of other
reducing agents, for example without limitation,
mercaptoethanol.
[0144] The spermidine and/or spermine concentration can range from
0 to 20 mM.
[0145] The PEG-8000 concentration can range from 0 to 50% (w/v).
The methods of the present invention also provide for the use of
other hydrophilic polymer, for example without limitation, other
molecular weight PEG or other polyalkylene glycols.
[0146] The Triton X-100 concentration can range from 0 to 0.1%
(w/v). The methods of the present invention also provide for the
use of other non-ionic detergents, for example without limitation,
other detergents, including other Triton-X detergents.
[0147] The MgCl.sub.2 concentration can range from 0.5 mM to 50 mM.
The MnCl.sub.2 concentration can range from 0.15 mM to 15 mM. Both
MgCl.sub.2 and MnCl.sub.2 must be present within the ranges
described and in a preferred embodiment are present in about a 10
to about 3 ratio of MgCl.sub.2:MnCl.sub.2, preferably, the ratio is
about 3-5, more preferably, the ratio is about 3 to about 4.
[0148] The 2'-OMe NTP concentration (each NTP) can range from 5
.mu.M to 5 mM.
[0149] The 2'-OH GTP concentration can range from 0 .mu.M to 300
.mu.M.
[0150] The 2'-OH GMP concentration can range from 0 to 5 mM.
[0151] The pH can range from pH 6 to pH 9. The methods of the
present invention can be practiced within the pH range of activity
of most polymerases that incorporate modified nucleotides.
[0152] In addition, the methods of the present invention provide
for the optional use of chelating agents in the transcription
reaction condition, for example without limitation, EDTA, EGTA, and
DTT.
[0153] Pharmaceutical Compositions
[0154] The invention also includes pharmaceutical compositions
containing the aptamer molecules described herein. In some
embodiments, the compositions are suitable for internal use and
include an effective amount of a pharmacologically active compound
of the invention, alone or in combination, with one or more
pharmaceutically acceptable carriers. The compounds are especially
useful in that they have very low, if any toxicity.
[0155] Compositions of the invention can be used to treat or
prevent a pathology, such as a disease or disorder, or alleviate
the symptoms of such disease or disorder in a patient. Compositions
of the invention are useful for administration to a subject
suffering from, or predisposed to, a disease or disorder which is
related to or derived from a target to which the aptamers
specifically bind.
[0156] For example, the target is a protein involved with a
pathology, for example, the target protein causes the
pathology.
[0157] Compositions of the invention can be used in a method for
treating a patient having a pathology. The method involves
administering to the patient a composition comprising aptamers that
bind a target (e.g., a protein) involved with the pathology, so
that binding of the composition to the target alters the biological
function of the target, thereby treating the pathology.
[0158] The patient having a pathology, e.g. the patient treated by
the methods of this invention can be a mammal, or more
particularly, a human.
[0159] In practice, the compounds or their pharmaceutically
acceptable salts, are administered in amounts which will be
sufficient to exert their desired biological activity.
[0160] For instance, for oral administration in the form of a
tablet or capsule (e.g., a gelatin capsule), the active drug
component can be combined with an oral, non-toxic pharmaceutically
acceptable inert carrier such as ethanol, glycerol, water and the
like. Moreover, when desired or necessary, suitable binders,
lubricants, disintegrating agents and coloring agents can also be
incorporated into the mixture. Suitable binders include starch,
magnesium aluminum silicate, starch paste, gelatin,
methylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone, natural sugars such as glucose or
beta-lactose, corn sweeteners, natural and synthetic gums such as
acacia, tragacanth or sodium alginate, polyethylene glycol, waxes
and the like. Lubricants used in these dosage forms include sodium
oleate, sodium stearate, magnesium stearate, sodium benzoate,
sodium acetate, sodium chloride, silica, talcum, stearic acid, its
magnesium or calcium salt and/or polyethyleneglycol and the like.
Disintegrators include, without limitation, starch, methyl
cellulose, agar, bentonite, xanthan gum starches, agar, alginic
acid or its sodium salt, or effervescent mixtures, and the like.
Diluents, include, e.g., lactose, dextrose, sucrose, mannitol,
sorbitol, cellulose and/or glycine.
[0161] Injectable compositions are preferably aqueous isotonic
solutions or suspensions, and suppositories are advantageously
prepared from fatty emulsions or suspensions. The compositions may
be sterilized and/or contain adjuvants, such as preserving,
stabilizing, wetting or emulsifying agents, solution promoters,
salts for regulating the osmotic pressure and/or buffers. In
addition, they may also contain other therapeutically valuable
substances. The compositions are prepared according to conventional
mixing, granulating or coating methods, respectively, and contain
about 0.1 to 75%, preferably about 1 to 50%, of the active
ingredient.
[0162] The compounds of the invention can also be administered in
such oral dosage forms as timed release and sustained release
tablets or capsules, pills, powders, granules, elixirs, tinctures,
suspensions, syrups and emulsions.
[0163] Liquid, particularly injectable compositions can, for
example, be prepared by dissolving, dispersing, etc. The active
compound is dissolved in or mixed with a pharmaceutically pure
solvent such as, for example, water, saline, aqueous dextrose,
glycerol, ethanol, and the like, to thereby form the injectable
solution or suspension. Additionally, solid forms suitable for
dissolving in liquid prior to injection can be formulated.
Injectable compositions are preferably aqueous isotonic solutions
or suspensions. The compositions may be sterilized and/or contain
adjuvants, such as preserving, stabilizing, wetting or emulsifying
agents, solution promoters, salts for regulating the osmotic
pressure and/or buffers. In addition, they may also contain other
therapeutically valuable substances.
[0164] The compounds of the present invention can be administered
in intravenous (both bolus and infusion), intraperitoneal,
subcutaneous or intramuscular form, all using forms well known to
those of ordinary skill in the pharmaceutical arts. Injectables can
be prepared in conventional forms, either as liquid solutions or
suspensions.
[0165] Parental injectable administration is generally used for
subcutaneous, intramuscular or intravenous injections and
infusions. Additionally, one approach for parenteral administration
employs the implantation of a slow-release or sustained-released
systems, which assures that a constant level of dosage is
maintained, according to U.S. Pat. No. 3,710,795, incorporated
herein by reference.
[0166] Furthermore, preferred compounds for the present invention
can be administered in intranasal form via topical use of suitable
intranasal vehicles, or via transdermal routes, using those forms
of transdermal skin patches well known to those of ordinary skill
in that art. To be administered in the form of a transdermal
delivery system, the dosage administration will, of course, be
continuous rather than intermittent throughout the dosage regimen.
Other preferred topical preparations include creams, ointments,
lotions, aerosol sprays and gels, wherein the concentration of
active ingredient would range from 0.01% to 15%, w/w or w/v.
[0167] For solid compositions, excipients include pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talcum, cellulose, glucose, sucrose, magnesium
carbonate, and the like may be used. The active compound defined
above, may be also formulated as suppositories using for example,
polyalkylene glycols, for example, propylene glycol, as the
carrier. In some embodiments, suppositories are advantageously
prepared from fatty emulsions or suspensions.
[0168] The compounds of the present invention can also be
administered in the form of liposome delivery systems, such as
small unilamellar vesicles, large unilamellar vesicles and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, containing cholesterol, stearylamine or
phosphatidylcholines. In some embodiments, a film of lipid
components is hydrated with an aqueous solution of drug to a form
lipid layer encapsulating the drug, as described in U.S. Pat. No.
5,262,564. For example, the aptamer molecules described herein can
be provided as a complex with a lipophilic compound or
non-immunogenic, high molecular weight compound constructed using
methods known in the art. An example of nucleic-acid associated
complexes is provided in U.S. Pat. No. 6,011,020.
[0169] The compounds of the present invention may also be coupled
with soluble polymers as targetable drug carriers. Such polymers
can include polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropyl-methacrylamide-p- henol,
polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine
substituted with palmitoyl residues. Furthermore, the compounds of
the present invention may be coupled to a class of biodegradable
polymers useful in achieving controlled release of a drug, for
example, polylactic acid, polyepsilon caprolactone, polyhydroxy
butyric acid, polyorthoesters, polyacetals, polydihydropyrans,
polycyanoacrylates and cross-linked or amphipathic block copolymers
of hydrogels.
[0170] If desired, the pharmaceutical composition to be
administered may also contain minor amounts of non-toxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, and other substances such as for example, sodium acetate,
triethanolamine, oleate, etc.
[0171] The dosage regimen utilizing the compounds is selected in
accordance with a variety of factors including type, species, age,
weight, sex and medical condition of the patient; the severity of
the condition to be treated; the route of administration; the renal
and hepatic function of the patient; and the particular compound or
salt thereof employed. An ordinarily skilled physician or
veterinarian can readily determine and prescribe the effective
amount of the drug required to prevent, counter or arrest the
progress of the condition.
[0172] Oral dosages of the present invention, when used for the
indicated effects, will range between about 0.05 to 1000 mg/day
orally. The compositions are preferably provided in the form of
scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0,
50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient.
Effective plasma levels of the compounds of the present invention
range from 0.002 mg to 50 mg per kg of body weight per day.
[0173] Compounds of the present invention may be administered in a
single daily dose, or the total daily dosage may be administered in
divided doses of two, three or four times daily.
[0174] All publications and patent documents cited herein are
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference. Citation of publications and
patent documents is not intended as an admission that any is
pertinent prior art, nor does it constitute any admission as to the
contents or date of the same.
[0175] The invention having now been described by way of written
description, those of skill in the art will recognize that the
invention can be practiced in a variety of embodiments and that the
foregoing description and examples below are for purposes of
illustration and not limitation of the claims that follow.
EXAMPLES
Example 1
2'-OMe SELEX.TM. Against Thrombin and VEGF Targets
[0176] A library of approximately 3.times.10.sup.14 unique
transcription templates, each containing a random region of thirty
contiguous nucleotides, was synthesized as described below, and PCR
amplified. Cloning and sequencing of this library demonstrated that
the composition of the random region in this library was
approximately 25% of each nucleotide. The DNA library was purified
away from unincorporated dNTPs by gel-filtration and
ethanol-precipitation. Modified transcripts were then generated
from a mixture containing 500 uM of each of the four 2'-OMe NTPs,
i.e., A, C, U and G, and 30 uM 2'-OH GTP ("r/mGmH"). In addition,
modified transcripts were generated from mixtures containing part
modified nucleotides and part ribonucleotides or all
ribonucleotides namely, a mixture containing all 2'-OH nucleotides
(rN); a mixture containing 2'-OMe C and U and 2'-OH A and G (rRmY);
a mixture containing 2'-OMe A, C, and U, and 2'-OH G ("rGmH"); and
a mixture alternately containing 2'-OMe A, C, U and G and 2'-OMe A,
U and C and 2'-F G ("toggle"). These modified transcripts were then
used in SELEX.TM. against targets--e.g., VEGF and thrombin.
[0177] Generally, after gel-purification and DNase-treatment these
modified transcripts were dissolved in PBS for VEGF or 1.times.ASB
(150 mM KCl, 20 mM HEPES, 10 mM MgCl.sub.2, 1 mM DTT, 0.05%
Tween20, pH 7.4) for thrombin, and incubated for one hour in an
empty well on a hydrophobic multiwell plate to subtract
plastic-binding sequences. The supernatant was then transferred to
a well that had previously been incubated for one hour at room
temperature in PBS for VEGF or in ASBND (150 mM KCl, 20 mM HEPES,
10 mM MgCl.sub.2, 1 mM DTT, pH 7.4) for thrombin. After a one hour
incubation the well was washed and bound sequences were
reverse-transcribed in situ using thermoscript reverse
transcriptase (Invitrogen) at 65.degree. C. for one hour. The
resultant cDNA was then PCR-amplified, separated from dNTPs by
gel-filtration, and used to generate modified transcripts for input
into the next round of selection. After 10 rounds of selection and
amplification the ability of the resultant library to bind to VEGF
or thrombin was assessed by Dot-Blot. At this point, the library
was cloned, sequenced and individual clones were assayed for their
ability to bind VEGF or thrombin. Using this combination of
sequence and clonal binding data, sequence motifs were
identified.
[0178] One VEGF aptamer motif, exemplified by ARC224, which was
common to both the r/mGmH and toggle selections, was used to design
smaller synthetic constructs which were also assayed for binding to
VEGF and ultimately minimized aptamers to VEGF were identified,
ARC245 and ARC259, both of which are 23 nucleotides long. Another
VEGF aptamer motif, exemplified by ARC226, which was common to all
2'-OMe selections, was also identified. The ARC224 aptamer produced
by the methods of the present invention has the sequence
5'-mCmGmAmUmAmUmGmCmAmGmUmUmUmGmAmGmAm- AmGmUmCmGmCmGmC
mAmUmUmCmG-3T (SEQ ID No. 184) where "m" represents a 2'-O-methyl
substitution.
[0179] The ARC226 aptamer has the sequence:
2 5-mGmAmUmCmAmUmGmCmAmUGmUmGmGmAmUm (SEQ ID No. 186)
CmGmCmGmGmAmUmC-[3T]-3'.
[0180] The ARC245 aptamer has sequence:
3 5'-mAmUmGmCmAmGmUmUmUmGmAmGmAmAmGm (SEQ ID No. 187)
UmCmGmCmGmCmAmU-[3T]-3'.
[0181] The ARC259 aptamer has the sequence:
4 5'-mAmCmGmCmAmGmUmUmUmGmAmGmAmAmGm (SEQ ID No. 188)
UmCmGmCmGmCmGMu-[3T]-3'.
[0182] FIG. 3A is a graph of VEGF binding by ARC224, ARC245 and
ARC259. A schematic representation of the secondary structure of
these aptamers is presented in FIG. 3B.
[0183] All residues in ARC224, ARC226 and ARC245 are 2'-OMe and all
constructs (initially identified by SELEX.TM.) were generated by
solid-phase chemical synthesis. The K.sub.D values of these
aptamers, determined by dot-blot in PBS, are as follows: ARC224 3.9
nM, ARC245 2.1 nM, ARC259 1.4 nM.
[0184] Reagents. All reagents were acquired from Sigma (St. Louis,
Mo.) except where otherwise stated.
[0185] Oligonucleotide synthesis. DNA syntheses were undertaken
according to standard protocols using an Expedite 8909 DNA
synthesizer (Applied Biosystems, Foster City, Calif.). The DNA
library used in this study had the following sequence: ARC254:
5'-CATCGATGCTAGTCGTAACGATCNCGAGAACGTTCTCT-
CCTCTCCCTATAGTGAGTCGTATTA-3' (SEQ ID NO:1) in which each N has an
equal probability of being each of the four nucleotides. 2'-OMe RNA
syntheses, including those containing 2'-OH nucleotides, were
undertaken according to standard protocols using a 3900 DNA
Synthesizer (Applied Biosystems, Foster City, Calif.). All
oligonucleotides were purified by denaturing PAGE except PCR and RT
primers.
[0186] 2'-OMe Library Generation. The synthetic DNA library (1.5
mmol) was amplified by PCR under standard conditions with the
following primers:
5 3'-primer 5'-CATCGATGCTAGTCGTAACGATCC-3' (SEQ ID NO:454) and
5'-primer 5'-TAATACGACTCACTATAGGGAGAGGA- GAGAA (SEQ ID NO:455)
ACGTTCTCG-3'.
[0187] The resultant library of double-stranded transcription
templates was precipitated and separated from unincorporated
nucleotides by gel-filtration. At no point was the library
denatured, either by thermal means or by exposure to low-salt
conditions. r/mGmH transcription was performed under the following
conditions to produce template for the first round of selection:
double-stranded DNA template 200 nM, HEPES 200 mM, DTT 40 mM,
Triton X-100 0.01%, Spermidine 2 mM, 2'-O-methyl ATP, CTP, GTP and
UTP 500 .mu.M each, 2'-OH GTP 30 uM, GMP 500 .mu.M, MgCl.sub.2 5.0
mM, MnCl.sub.2 1.5 mM, inorganic pyrophosphatase 0.5 units per 100
.mu.L reaction, Y639F/H784A T7 RNA polymerase 1.5 units per 100
.mu.l reaction pH 7.5 and 10% w/v PEG and were incubated at
37.degree. C. overnight. The resultant transcripts were purified by
denaturing 10% PAGE, eluted from the gel, incubated with RQ1 DNase
(Promega, Madison Wis.), phenol-extracted, chloroform-extracted,
precipitated and taken up in PBS. For the initiation of selection
transcripts were additionally generated by the direct chemical
synthesis of 2'-OMe RNA, these were purified by denaturing 10%
polyacrylamide gel electrophoresis, eluted from the gel and taken
up in PBS.
[0188] For the rN, rRmY and rGmH transcriptions, the transcription
conditions were as follows, where 1.times.Tc buffer is: 200 mM
HEPES, 40 mM DTT, 2 mM Spermidine, 0.01% Triton X-100, pH 7.5.
[0189] When 2'-OH A, C, U and G (rN) conditions were used, the
transcription reaction conditions were MgCl.sub.2 25 mM, each NTP 5
mM, 1.times.Tc buffer, 10% w/v PEG, T7 RNA polymerase 1.5 units,
and 50-200 nM double stranded template (200 nM of template was used
in Round 1 to increase diversity and for subsequent rounds
approximately 50 nM, a {fraction (1/10)} dilution of an optimized
PCR reaction using conditions described herein, was used).
[0190] When 2'-OMe C and U and 2'-OH A and G (rRmY) conditions were
used, the transcription reaction conditions were 1.times.Tc buffer,
50-200 nM double stranded template (200 nM of template was used in
Round 1 to increase diversity and for subsequent rounds
approximately 50 nM, a {fraction (1/10)} dilution of an optimized
PCR reaction using conditions described herein, was used), 5.0 mM
MgCl.sub.2, 1.5 mM MnCl.sub.2, 0.5 mM each base, 10% PEG-8000, 0.25
units inorganic pyrophosphatase, and 1.5 units Y639F/H784A T7 RNA
polymerase.
[0191] When 2'-OMe A, C, and U and 2'-OH G (rGmH) conditions were
used, the transcription reaction conditions were 1.times.Tc buffer,
50-200 nM double stranded DNA template (200 nM of template was used
in Round 1 to increase diversity for subsequent rounds
approximately 50 nM, a {fraction (1/10)} dilution of an optimized
PCR reaction using conditions described herein, was used), 5.0 mM
MgCl.sub.2, 1.5 mM MnCl.sub.2, 0.5 mM each base, 10% PEG-8000, 0.25
units inorganic pyrophosphatase, and 1.5 units Y639F single mutant
T7 RNA polymerase in 100 .mu.l volume.
[0192] When 2'-OMe A, C, U and 2'-F G conditions were used, the
transcription reaction conditions were as for rGmH, except 0.5 mM
2'-F GTP is used instead of 2'-OH GTP.
[0193] Reverse Transcription. The reverse transcription conditions
used during SELEX.TM. are as follows (100 .mu.L reaction volume):
1.times. Thermo buffer (Invitrogen), 4 .mu.M primer, 10 mM DTT, 0.2
mM each dNTP, 200 .mu.M Vanadate nucleotide inhibitor, 10 .mu.g/ml
tRNA, Thermoscript RT enzyme 1.5 units (Invitrogen). Reverse
transcriptase reaction yields are lower for 2'-OMe templates. PCR
reaction conditions are as follows 1.times. ThermoPol buffer (NEB),
0.5 .mu.M 5' primer, 0.5 .mu.M 3' primer 0.2 mM each DHTP, Taq DNA
Polymerase 5 units (NEB).
[0194] 2'-OMe SELEX.TM. Protocol. As noted above, SELEX.TM. was
performed with the modified transcripts against each of two targets
(VEGF and Thrombin) using 5 kinds of transcripts for a total of 10
selections. The five kinds of transcripts were: "rN" (all 2'-OH),
"rRmY" (2'-OH A, G, 2'-OMe C, U), "rGmH" (2'-OH G, 2'-OMe C, U, A),
"r/mGmH" (2'-OMe A, U, G, C 500 uM, 2'-OH G 30 uM), "toggle"
(alternately "r/mGmH" and 2'-OMe A, U, C, 2'-F G).
[0195] All of the selections directed against VEGF generated VEGF
specific aptamers while only the rN and rRmY selections against
thrombin generated thrombin specific aptamers. The aptamer
sequences identified in these selections are set forth in Tables 1
through 5 (VEGF) and Tables 6 through 10 (thrombin) below.
[0196] The sequences are from SELEX.TM. round 11 except for
Thrombin "rGmH", "r/mGmH" and "toggle" which are from round 5, VEGF
"r/mGmH" which is from round 10 and VEGF "toggle" which is from
round 8.
[0197] The selection was performed by initially immobilizing the
protein by hydrophobic absorption to "NUNC MAXY" plates, washing
away the protein that didn't bind, incubating the library of
2'-OMe-substituted transcripts with the immobilized protein,
washing away the transcripts that didn't bind, performing RT
directly in the plate, then PCR, and then transcribing the
resultant double-stranded DNA template under the appropriate
transcription conditions.
[0198] Binding assays were performed with trace
.sup.32P-body-labelled transcripts that were incubated with various
protein concentrations in silanized wells, these were then passed
through a sandwich of a nitrocellulose membrane over a nylon
membrane. Protein-bound RNA is visualized on the NC membrane,
unbound RNA on the nylon membrane. The proportion binding is then
used to calculate affinity (see FIGS. 4, 5, and 6). For example,
the binding characteristics of various 2'-OH G variants of ARC224
(all 2-OMe) are shown in FIG. 4. The nomenclature "mGXG" indicates
a substitution of 2'-OH G for 2'-OMe G at position "X", as numbered
sequentially from the 5'-terminus. Thus, mG7G ARC224 is ARC224 with
a 2'-OH at position 7. ARC225 is ARC224 with 2'-OMe to 2'-OH
substitutions at positions 7, 10, 14, 16, 19, 22 and 24. All
constructs (initially identified by SELEX.TM.) were generated by
solid-phase chemical synthesis. These data were generated by
dot-blot in PBS. The fully 2'-OMe aptamer, ARC224, has superior
VEGF-binding characteristics when compared to any of the 2'-OH
substituted variants studied.
[0199] FIG. 5 is a plot of ARC224 and ARC225 binding to VEGF. This
graph indicates that ARC224 binds VEGF in a manner which inhibits
the biological function of VEGF. .sup.12I-labeled VEGF was
incubated with the aptamer and this mixture was then incubated with
human umbilical cord vascular endothelial cells (HUVEC). The
supernatant was removed, the cells were washed, and bound VEGF was
counted in a scintillation counter. ARC225 has the same sequence as
ARC224 and 2'-OMe to 2'-OH substitutions at positions 7, 10, 14,
16, 19, 22 and 24 numbered from the 5'-terminus. These data
indicate that the IC.sub.50 of ARC224 is approximately 2 nM.
[0200] FIG. 6 is a binding curve plot of ARC224 binding to VEGF
before and after autoclaving, with or without EDTA. FIG. 6 shows
both the proportion of aptamer that is functional and the IC.sub.50
for binding to VEGF before and after autoclaving for 25 minutes
with a peak temperature of 125.degree. C. These data were
determined by the inhibition by unlabeled ARC224 of the binding of
5'-labeled ARC224 to 1 nM VEGF in PBS as measured by dot-blot in
PBS. Where indicated, samples contained 1 mM EDTA. All constructs
(initially identified by SELEX.TM.) were generated by solid-phase
chemical synthesis. No degradation of ARC224 was observed within
the limitations of this assay.
[0201] Degradation studies show that incubation in plasma at
37.degree. C. over 4 days induces so little degradation that
measuring a half-life is not possible, but is at least in excess of
4 days (see, e.g., FIG. 7). FIGS. 7A and 7B are plots of the
stability of ARC224 and ARC226, respectively, when incubated at
37.degree. C. in rat plasma. As indicated in the figure, both
ARC224 and ACR226 showed no detectable degradation after for 4 days
in rat plasma. In these experiments, 5'-labeled ARC224 and ARC226
were incubated in rat plasma at 37.degree. C. and analyzed by
denaturing PAGE. All constructs (initially identified by SELEX.TM.)
were generated by solid-phase chemical synthesis. The half-life
appears to be in excess of 100 hours.
[0202] Tables 1 through Table 10 below show the DNA sequences of
aptamers corresponding to the transcribed aptamers isolated from
the various libraries, i.e. rN, rRmY, rGmH, and r/mGmH, as
indicated. The sequence of the aptamers will have uridine residues
instead of thymidine residues in the DNA sequences shown. Table 11
shows the stabilized aptamer sequences obtained by the methods of
the present invention. As used herein, "3T" refers to an inverted
thymidine nucleotide attached to the oligonucleotide phosphodiester
backbone at the 5' position, the resulting oligo having two 5'-OH
ends and is thus resistant to 3' nucleases.
[0203] Unless noted otherwise, individual sequences listed in the
various tables represent the cDNA clones of the aptamers that were
selected under the SELEX conditions provided. The actual aptamers
provided in the invention are those corresponding sequences
comprising the rN, mN, rRmY, rGmH, r/mGmH, dRmY and toggle
combinations of residues, as indicated in the text.
[0204] 2'-OMe SELEX.TM. Results.
6TABLE 1 Corresponding cDNAs of the VEGF Aptamer Sequences - all
2'-OH (rN) SEQ ID No. 3 >PB.97.126.F_43-H1
GGGAGAGGAGAGAACGTTCTCGAAATGATGCATGTTCGTAAAA- TGGCAGT
ATTGGATCGTTACAACTAGCATCGATG SEQ ID No. 4 >PB.97.126.F_43-A2
GGGAGAGGAGAGAACGTTCTCGTGCCGAGGTCCGGAACCTTGAT- GATTGG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 5 >PB.97.126.F_48-A1
GGGAGAGGAGAGAACGTTCTCGCATTTGGGCTAGTTGTGAAATG- GCAGTA
TTGGATCGTTACGACTAGCATCGATG SEQ ID No. 6 >PB.97.126.F_48-B1
GGGAGAGGAGAGAACGTTCTCGAATCGTAGATAGTCGTGAAATG- GCAGTA
TTGGATCGTTACGACTAGCATCGATG SEQ ID No. 7 >PB.97.126.F_48-C1
GGGAGAGGAGAGAACGTTCTCGTTCTAGTCGGTACGATATGTTG- ACGAAT
CCGGATCGTTACGACTAGCATCGATG SEQ ID No. 8 >PB.97.126.F_48-D1
GGGAGAGGAGAGAACGTTCTCGTTTGATGAGGCGGACATAATCC- GTGCCG
AGCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 9 >PB.97.126.F_48-E1
GGGAGAGGAGAGAACGTTCTCGAAGGAAAAGAGTTTAGTATTGG- CCGTCC
GTGGGATCGTTACGACTAGCATCGATG SEQ ID No. 10 >PB.97.126.F_48-F1
GGGAGAGGAGAGAACGTTCTCGTGCCGAGGTCCGGAACCTTGAT- GATTGG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 11 >PB.97.126.F_48-G1
GGGAGAGGAGAGAACGTTCTCGTACGGTCCATTGAGTTTGAGAT- GTCGCC
ATGGATCGTTACGACTAGCATCGATG SEQ ID No. 12 >PB.97.126.F_48-B2
GGGAGAGGAGAGAACGTTCTCGAGTTAGTGGTAACTGATATGTT- GAATTG
TCCGGATCGTTACGACTAGCATCGATG SEQ ID No. 13 >PB.97.126.F_48-C2
GGGAGAGGAGAGAACGTTCTCGCACGGATGGCGAGAACAGAGAT- TGCTAG
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 14 >PB.97.126.F_48-D2
GGGAGAGGAGAGAACGTTCTCGNTANCGNTNCGCCNTGCTAACG- CNTANT
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 15 >PB.97.126.F_48-E2
GGGAGAGGAGAGAACGTTCTCGAAGATGAGTTTTGTCGTGAAAT- GGCAGT
ATTGGATCGTTACGACTAGCATCGATG SEQ ID No. 16 >PB.97.126.F_48-F2
GGGAGAGGAGAGAACGTTCTCGGGATGCCGGATTGATTTCTGAT- GGGTAC
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 17 >PB.97.126.F_48-G2
GGGAGAGGAGAGAACGTTCTCGAATGGAATGCATGTCCATCGCT- AGCATT
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 18 >PB.97.126.F_48-H2
GGGAGAGGAGAGAACGTTCTCGTGCTGAGGTCCGGAACCTTGAT- GATTGG
CGGGATCGTTNCNACTAGCATCGATG SEQ ID No. 19 >PB.97.126.F_48-A3
GGGAGAGGAGAGAACGTTCTCGCTAATTGCTGAGTCGTGAAGTG- GCAGTA
TTGGATCGTTACGACTAGCATCGATG SEQ ID No. 20 >PB.97.126.F_48-B3
GGGAGAGGAGAGAACGTTCTCGTAACGATGTCCGGGGCGAAAGG- CTAGCA
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 21 >PB.97.126.F_48-C3
GGGAGAGGAGAGAACGTTCTCGATGCGATTGTCGAGATTTGTAA- GATAGC
TGTGGATCGTTACGACTAGCATCGATG
[0205]
7TABLE 2 Corresponding cDNAs of the VEGF Aptamer Sequences - 2'-OH
AG, 2'-OMe CU (rRmY) SEQ ID No. 22 >PB.97.126.G_43-D3
GGGAGAGGAGAGAACGTTCTCGCAGAAAACATCT- TTGCGGTTGAATACAT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 23 >PB.97.126.G_43-G3
GGGAGAGGAGAGAACGTTCTCGAAAAAAGANANCN- NCCTTCNGAATACAT
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 24 >PB.97.126.G_48-E3
GGGAGAGGAGAGAACGTTCTCGAGAGTGATTCGAT- GCTTCANGAATACAT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 25 >PB.97.126.G_48-F3
GGGAGAGGAGAGAACGTTCTCGAGAGTGATTCGAT- GCTTCANGAATACAT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 26 >PB.97.126.G_48-H3
GGGAGAGGAGAGAACGTTCTCGAAGAAGGAAAGCT- GCAAGTCGAATACAC
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 27 >PB.97.126.G_48-A4
GGGAGAGGAGAGAACGTTCTCGCAAAAACATCGAT- TACAGTTGAGTACAT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 28 >PB.97.126.G_48-B4
GGGAGAGGAGAGAACGTTCTCGAGACATCATTGCT- CGTTGAATACATGTG
GATCGTTACGACTAGCATCGATG SEQ ID No. 29 >PB.97.126.G_48-C4
GGGAGAGGAGAGAACGTTCTCGCCAAAGTAGCTTCGACA- GTCGAATACAT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 30 >PB.97.126.G_48-D4
GGGAGAGGAGAGAACGTTCTCGAAAATCAGTACTGTGCA- GTCGAATACAT
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 31 >PB.97.126.G_48-E4
GGGAGAGGAGAGAACGTTCTCGTAATGACATCAATGCTT- CTTGAATACAG
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 32 >PB.97.126.G_48-F4
GGGAGAGGAGAGAACGTTCTCGAGAAAAACGATCTGTGA- CGTGTAATCCG
CGGATCGTTACGACTAGCATCGATG SEQ ID No. 33 >PB.97.126.G_48-G4
GGGAGAGGAGAGAACGTTCTCGCAACAAACGTCGACGCTTCT- GAATACAT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 34 >PB.97.126.G_48-H4
GGGAGAGGAGAGAACGTTCTCGTGATCATAGAAATGCTAGCTGA- ATACAT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 35 >PB.97.126.G_48-A5
GGGAGAGGAGAGAACGTTCTCGCAGCGTAAAATGCTTTTCGAAG- TACATG
TGGATCGTTACGACTAGCATCGATG SEQ ID No. 36 SEQ ID No.
>PB.97.126.G_48-B5 GGGAGAGGAGAGAACGTTCTCGCCAAGAATC-
AATCGCTTGTCGAATACAT GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 37
>PB.97.126.G_48-C5 GGGAGAGGAGAGAACGTTCTCGTGATCATAGA-
AATGCTAGCTGAGTACAT GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 38
>PB.97.126.G_48-D5 GGGAGAGGAGAGAACGTTCTCGCAGAAAACAT-
CTTTGCGGTTGAATACAT GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 39
>PB.97.126.G_48-E5 GGGAGAGGAGAGAACGTTCTCGNAAACANNCA-
TCTATTGNAGTTGAATAC ATGTGGATCGTTACGACTAGCATCGATG SEQ ID No. 40
>PB.97.126.G_48-F5 GGGAGAGGAGAGAACGTTCTCGCTAAAGATTC-
GCTGCTTGCCGAATACAT GTGGATCGTTACGACTAGCATCGATG
[0206]
8TABLE 3 Corresponding cDNAs of the VEGF Aptamer Sequences - 2'-OH
G, 2'-OMe CUA (rGmH) SEQ ID No. 41 >PB.97.126.H_43-H6
GGGAGAGGAGAGAACGTTCTCGGGTTTTGTCTGC- GTTTGTGCGTTGAACC
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 42 >PB.97.126.H_43-F7
GGGAGAGGAGAGAACGTTCTCGTGATTACGTGATG- AGGATCCGCGTTTTC
TCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 43 >PB.97.126.H_43-H7
GGGAGAGGAGAGAACGTTCTCGTTAGTGAAAACGA- TCATGCATGTGGATC
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 44 >PB.97.126.H_48-H5
GGGAGAGGAGAGAACGTTCTCGTGTTCATTCGTTT- GCTTATCGTTGCATG
TGGATCGTTACGACTAGCATCGATG SEQ ID No. 45 >PB.97.126.H_48-A6
AGGAGAGGAGAGAACGTTCTCGGCAGAGTGTGATG- TGCATCCGCACGTGC
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 46 >PB.97.126.H_48-B6
GGGAGAGGAGAGAACGTTCTCGTTAGTAAATACGA- TCGTGCATGTGGATC
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 47 >PB.97.126.H_48-C6
GGGAGAGGAGAGAACGCCCCCCTGATTNCGTGAAG- AGGATCCGCANTTTC
NCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 48 >PB.97.126.H_48-D6
GGGAGAGGAGAGAACGTTCTCGTGGCTTTGGAACG- GGTACGGATTTGGCA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 49 >PB.97.126.H_48-E6
GGGAGAGGAGAGAACGTTCTCGTGATTACGTGATG- AGGATCCGCGTTTTC
TCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 50 >PB.97.126.H_48-F6
GGGAGAGGAGAGAACGTTCTCGTCATTGGTGACNG- CGTTGCATGTGGATC
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 51 >PB.97.126.H_48-G6
GGGAGAGGAGAGAACGTTCTCGNTGGTNNAANGCT- TTTGTNGGGNTANNT
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 52 SEQ ID No.
>PB.97.126.H_48-A7
GGGAGAGGAGAGAACGTTCTCGTGGCTTTGGAACGAATTCGGATTTGGCA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 53 >PB.97.126.H_48-B7
GGGAGAGGAGAGAACGTTCTCGTGCGATGTCGTGGATTTCCGTT- TCGCAA
GGGATCGTTACGACTAGCATCGATG SEQ ID No. 54 PB.97.126.H_48-C7
GGGAGAGGAGAGAACGTTCTCGTGAAGCAGATGTCGTTGGCGACTTAG- AG
GGGGATCGTTACGACTAGCATCGATG SEQ ID No. 55 >PB.97.126.H_48-D7
GGGAGAGGAGAGAACGTTCTCGTGATTTCGTGATGAGGATCCGC- GTTTTC
TCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 56 >PB.97.126.H_48-E7
GGGAGAGGAGAGAACGTTCTCGCTAGTAACGATGACTTGATGAG- CATCCG
AGGATCGTTACGACTAGCATCGATG SEQ ID No. 57 >PB.97.126.H_48-G7
GGGAGAGGAGAGAACGTTCTCGTCATAAGTAACGACGTTGCATG- TGGATC
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 58 >PB.97.126.H_48-A8
GGGAGAGGAGAGAACGTTCTCGCAAGGAGATGGTTGCTAGCTGA- GTACAT
GTGGATCGTTACGACTAGCATCGATG
[0207]
9TABLE 4 Corresponding cDNAs of the VEGF Aptamer Sequences - 2'-OMe
AUGC (r/mGmH, each G has a 90% probability of having a 2'-OMe group
incorporated therein) SEQ ID No. 59 PB.97.126.I_43-B8
GGGAGAGGAGAGAACGTTCTCGCGATA- TGCAGTTTGAGAAGTCGCGCATT
CGGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 60 >PB.97.126.I_48-C8
GGGAGAGGAGAGAACGTTCTCGTGCGA- CGGGCTTCTTGTGTCATTCGCAT
GGGATCGTTACGACTAGCATCGATG SEQ ID No. 61 >PB.97.126.I_48-D8
GGGAGAGGAGAGAACGTTCTCGGCATTG- CAGTTGATAGGTCGCGCAGTGC
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 62 >PB.97.126.I_48-E8
GGGAGAGGAGAGAACGTTCTCGCGATAT- GCAGTCTGAGAAGTCGCGCATT
CGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 63 >PB.97.126.I_48-F8
GGGAGAGGAGAGAACGTTCTCGTGTAGC- AAGCATGTGGATCGCGACTGCA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 64 >PB.97.126.I_48-G8
GGGAGAGGAGAGAACGTTCTCGGATAAG- CAGTTGAGATGTCGCGCTTTGA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 65 >PB.97.126.I_48-H8
GGGAGAGGAGAGAACGTTCTCGATGANC- ANTTTGAGAAGTCGCGCTTGTC
GGGATCGTTACGACTAGCATCGATG SEQ ID No. 66 >PB.97.126.I_48-A9
GGGAGAGGAGAGAACGTTCTCGAGTAAT- GCAGTGGAAGTCGCGCATTACC
TGGGATCGTTACGACTAGCATCATG SEQ ID No. 67 >PB.97.126.I_48-B9
GGGAGAGGAGAGAACGTTCTCGCGATAT- GCAGTTTGAGAAGTCGCGCATT
CGGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 68 >PB.97.126.I_48-C9
GGGAGAGGAGAGAACGTTCTCGTGATNC- AGTTGANAAGTCNCGCATACAG
GATCGTTACGACTAGCATCGATG SEQ ID No. 69 >PB.97.126.I_48-D9
GGGAGAGGAGAGAACGTTCTCGAGTAATGCTG- TGGAAGTCGCGCATTTCC
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 70 >PB.97.126.I_48-D8
GGGAGAGGAGAGAACGTTCTCGGCATTGCAGT- TGATAGGTCGCGCAGTGC
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 71 >PB.97.126.I_48-F9
GGGAGAGGAGAGAACGTTCTCGCGATATGCAG- TTTGGGAAGTCGCGCATT
CGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 72 >PB.97.126.I_48-G9
GGGAGAGGAGAGAACGTTCTCGCNATATGCTG- TTTGANAANTCGCGCATT
CGGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 73 >PB.97.126.I_48-H9
GGGAGAGGAGAGAACGTTCTCGCGTAGATTGG- GCTGAATGGGATATCTTT
AGCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 74 >PB.97.126.I_48-B10
GGGAGAGGAGAGAACGTTCTCGCGATATGCA- GTTTGAGAAGTCGCGCTTT
CGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 75 >PB.97.126.I_48-D10
GGGAGAGGAGAGAACGTTCTCGTCAAT- CTGATGTAGCCTCACGTGGGCGG
AGTCGGATCGTTACGACTAGCATCGATG
[0208]
10TABLE 5 Corresponding cDNAs of the VEGF Aptamer Sequences -
alternately "r/mGmH" and 2'-OMe AUC, 2'-F G (toggle) SEQ ID No. 76
>PB.97.126.J_48-F10
GGGAGAGGAGAGAACGTTCTCGGATCGTTACGACTAGCATCGATG SEQ ID No. 77
>PB.97.126.J_48-G10 GGGAGAGGAGAGAACGTTCTCGGATCGTTACGAC-
TAGCATCGATG SEQ ID No. 78 >PB.97.126.J_48-H10
GGGAGAGGAGAGAACGTTCTCGGTGGTGTTGCTGAACTGTCGCGTTTCGC
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 79 >PB.97.126.J_48-A11
GGGAGAGGAGAGAACGTTCTCGTCGCGATTGCATATTTTCCGC- CTTGCTG
TGAGGATCGTTACGACTAGCATCGATG SEQ ID No. 80 >PB.97.126.J_48-B11
GGGAGAGGAGAGAACGTTCTCGCGATTTGCAGTTTGAGATGTC- GCGCATT
CGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 81 >PB.97.126.J_48-C11
GGGAGAGGAGAGAACGTTCTCGCGATATGCAGTTTGAGAAGT- CGCGCATT
CGGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 82 >PB.97.126.J_48-D11
GGGAGAGGAGAGAACGTTCTCGTTGGTGCAGTTTGAGATGT- CGCGCACCT
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 83 >PB.97.126.J_48-E11
GGGAGAGGAGAGAACGTTCTCGGTATTGGTTCCATTAAGCTG- GACACTCT
GCTCCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 84 >PB.97.126.J_48-F11
GGGAGAGGAGAGAACGTTCTCGTTGGTGCAGTTTGAGA- TGTCGCGCGCCT
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 85 >PB.97.126.J_48-G11
GGGAGAGGAGAGAACGTTCTCGCGATATGCAGTTTGAG- AAGTCGCGCATT
CGAGGGATCGTTACNACTAGCATCGATG SEQ ID No. 86 >PB.97.126.J_48-A12
GGGAGAGGAGAGAACGTTCTCGCGATATGCAGTT- TGAGAAGTCGCGCATT
CGGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 87 >PB.97.126.J_48-B12
GGGAGAGGAGAGAACGCTCTCGGGGACNNAA- ANNCGAATTGNCGCGTGNG
TCCGGGGGAGCGCCCGACTAGTCATCGATG SEQ ID No. 88 >PB.97.126.J_48-C12
GGGAGAGGAGAGAACGTTCTCGCGATA- TGNANTTTGAGAAGTCGCGCATT
CGGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 89 >PB.97.126.J_48-D12
GGGAGAGGAGAGAACGTTCTCGGTGT- ACAGCTTGAGATGTCGCGTACTCC
GGGATCGTTACGACTAGCATCGATG SEQ ID No. 90 >PB.97.126.J_48-E12
GGGAGAGGAGAGAACGTTCTCGCGATA- TGCAGTTTGAGAAGTCGCGCATT
CGGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 91 >PB.97.126.J_48-F12
GGGAGAGGAGAGAACGTTCTCGAGTA- AGAAAGCTGAATGGTCGCACTTCT
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 92 >PB.97.126.J_48-G12
AGGGAGAGGAAGAACGTTCTCGCGATG- TGCAGTTTGAGAAGTCGCGCATT
CGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 93 >PB.97.126.J_48-H12
GGGAGAGGAGAGAACGTTCTCGAAAG- AATCAGCATGCGGATCGCGGCTTT
CGGGATCGTTACGACTAGCATCGATG
[0209]
11TABLE 6 Corresponding cDNAs of the Thrombin Aptamer Sequences -
all 2'-OH (rN) SEQ ID No. 94 >PB.97.126.A_44-A1
GGGAGAGGAGAGAACGTTCTCGANTCCANTNTNC- NTGGAGGAGTAAGTAC
CTGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 95 >PB.97.126.A_44-B1
GGGAGAGGAGAGAACGTTCTCGGGAAACAAG- GAACTTAGAGTTANTTGAC
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 96 >PB.97.126.A_44-C1
GGGAGAGGAGAGAACGTTCTCGTACCATGCA- AGGAACATAATAGTTAGCG
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 97 >PB.97.126.A_44-D1
GGGAGAGGAGAGAACGTTCTCGGGACACAAG- GAACACAATAGTTAGTGTA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 98 >PB.97.126.A_44-E1
GGGAGAGGAGAGAACGTTCTCGTCTGCAAGG- AACACAATAGTTAGCATTG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 99 >PB.97.126.A_44-F1
GGGAGAGGAGAGAACGTTCTCGCGCCAACAA- AGCTGGAGTACTTAGAGCG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 100 PB.97.126.A_44-G1
GGGAGAGGAGAGAACGTTCTCGATTGCAAAATAGC- TGTAGAACTAAGCAA
TCGGATCGTTACGACTAGCATCGATG SEQ ID No. 101 >PB.97.126.A_44-H1
GGGAGAGGAGAGAACGTTCTCGTGAGATGACTAT- GTTAAGATGACGCTGT
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 102 >PB.97.126.A_44-A2
GGGAGAGGAGAGAACGTTCTCGGGANACAAGGAA- CNCAATATTTAGTGAA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 103 >PB.97.126.A_44-B2
GGGAGAGGAGAGAACGTTCTCGCCAAGGAACACA- ATAGTTAGGTGAGAAT
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 104 >PB.97.126.A_44-C2
GGGAGAGGAGAGACGTTCTCGGTACAAGGAACAC- AATAGTTAGTGCCGTG
GGATCGTTACGACTAGCATCGATG SEQ ID No. 105 >PB.97.126.A_44-D2
GGGAGAGGAGAGAACGTTCTCGATTCAACGGTCC- AAAAAAGCTGTAGTAC
TTAGGATCGTTACGACTAGCATCGATG SEQ ID No. 106 >PB.97.126.A_44-E2
GGGAGAGGAGAGAACGTTCTCGCAATGCAAGGAA- CACAATAGTTAGCAGC
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 107 >PB.97.126.A_44-F2
GGGAGAGGAGAGAACGTTCTCGAAAGGAGAAAGC- TGAAGTACTTACTATG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 108 >PB.97.126.A_44-G2
GGGAGAGGAGAGAACGTTCTCGCACAAGGAACAC- AATAGTTAGTGCAAGA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 109 >PB.97.126.A_44-A3
GGGAGAGGAGAGAACGTTCTCGCACAAGGAACTA- CGAGTTAGTGTGGGAG
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 110 >PB.97.126.A_44-B3
GGGAGAGGAGAGAACGTTCTCGCACAAGGAACAC- AATAGTTAGTGCAAGA
CGGGATCGTTACGACTAGCATCGATA SEQ ID No. 111 >PB.97.126.A_44-C3
GGGAGAGGAGAGAACGTTCTCGGCGGGAAAATAG- CTGTAGTACTAACCCA
CGGATCGTTACGACTAGCATCGATG
[0210]
12TABLE 7 Corresponding cDNAs of the Thrombin Aptamer Sequences -
2'-OH AG, 2'-OMe CU (rRmY) SEQ ID No. 112 >PB.97.126.B_44-E3
GGGAGAGGAGAGAACGTTCTCGGCCTCAAGGAAAAGAAAATTTAGAGGCC
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 113 >PB.97.126.B_44-F3
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGAAGGACTA- AGTTTA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 114 >PB.97.126.B_44-G3
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGAAGGACTA- AGTTTA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 115 >PB.97.126.B_44-H3
GGGAGAGGAGAGAACGTTCTCGGAGCCAAGGAAACGAAGATTTA- GGCTCA
TTGGGATCGTTACGACTAGCATCGATG SEQ ID No. 116 >PB.97.126.B_44-A4
GGGAGAGGAGAGAACGTTCTCGATCACAAGAAATGTGGGANGGT- AGTGAT
NCNNNTCGTTNCGACTAGCATCGATG SEQ ID No. 117 >PB.97.126.B_44-B4
GGGAGAGGAGAGAACGTTCTCGTCGAAAGGGAGCTTTGTCTCGG- GACAGA
ACGGATCGTTACGACTAGCATCGATG SEQ ID No. 118 >PB.97.126.B_44-C4
GGGAGAGGAGAGAACGNTCTCGTGCAAAGATAGCTGGAGGACTA- ATGCGG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 119 >PB.97.126.B_44-D4
GGGAGAGGAGAGAACGTTCTCGTCGAAAGGGAGCTTTGTCTCGG- GACAGA
ACGGATCGTTACGACTAGCATCGATG SEQ ID No. 120 >PB.97.126.B_44-E4
GGGAGAGGAGAGAACGTTCTCGNCNAAGGNGAGCTTTGTCCCNG- GACANA
ANGNATCGTTACAACTAGCATCGATG SEQ ID No. 121 >PB.97.126.B_44-F4
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGAAGGACTA- AGTTTA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 122 >PB.97.126.B_44-G4
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGAAGGACTA- AGTTTA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 123 >PB.97.126.B_44-H4
GGGAGAGGAGAGAACGTTCTCGGCGCAAAAAAAGCTGGAGTACT- TAGTGT
CGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 124 >PB.97.126.B_44-A5
GGGAGAGGAGAGAACGTTCTCGTCGAAAGGGAGCTTTGTCTCG- GGACAGA
ACGGATCGTTACGACTAGCATCGATG SEQ ID No. 125 >PB.97.126.B_44-B5
GGGAGAGGAGAGAACGTTCTCGACACAAGAAAGCTGCAGAACTT- AGGGTC
GTGGATCGTTACGACTAGCATCGATG SEQ ID No. 126 >PB.97.126.B_44-C5
GGGAGAGGAGAGAACGTTCTCGGAACNGGATTGTTGAAGGACTA- ANTTTA
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 127 >PB.97.126.B_44-D5
GGGAGAGGAGAGAACGTTCTCGGCCTCAAGGGAAAGAAAATTTA- GAGGCC
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 128 >PB.97.126.B_44-E5
GGGAGAGGAGAGAACGTTCTCGGAAACAAGCTTAGAAATTCGCA- CCCTTG
CCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 129 >PB.97.126.B_44-F5
GGGAGAGGAGAGAACGTTCTCGAAAGAAAAAAGCTGGAGAACTT- ACTTCC
GGGATCGTTACGACTAGCATCGATG SEQ ID No. 130 >PB.97.126.B_44-G5
GGGAGAGGAGAGAACGTTCTCGGTGATTGTACTCACATAGAAAT- GGCAAC
ACTGGGATCGTTACGACTAGCATCGATG
[0211]
13TABLE 8 Corresponding cDNAs of the Thrombin Aptamer Sequences -
2'-OH G, 2'-OMe CUA (rGmH) SEQ ID No. 132 >PB.97.126.C_44-H5
GGGAGAGGAGAGAACGTTCTCGGGTTCAAGGAACATGATAGTTAGAACCC
GCGGATCGTTACGACTAGCATCGATG SEQ ID No. 132 >PB.97.126.C_44-A6
GGGAGAGGAGAGAACGTTCTCGTTCCGAAAGGAACACAATAGTT- ATCGGA
TTGGGATCGTTACGACTAGCATCGATG SEQ ID No. 133 >PB.97.126.C_44-B6
GGGAGAGGAGAGACGTTCTCGTCTGCAAGGAACACAATAGTTAG- CATTGC
GGGATCGTTACGACTAGCATCGATG SEQ ID No. 134 >PB.97.126.C_44-C6
GGGAGAGGAGAGAACGTTCTCGGTACAAGGAACACAATAGTTAG- TGCCGG
GGATCGTTACGACTAGCATCGATG SEQ ID No. 135 >PB.97.126.C_44-D6
GGGAGAGGAGAGAACGTTCTCGGAACTCAGAGATCCTATGTGGA- CCAGAG
AGGATCGTTACGACTAGCATCGATG SEQ ID No. 136 >PB.97.126.C_44-E6
GGGAGAGGAGAGAACGTTCTCGCTGAGCAAGGAACGTAATAGTT- AGCCTG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 137 >PB.97.126.C_44-F6
GGGAGAGGAGAGAACGTTCTCGNANNNATAAATGATGGATCNCT- TATTG
TNNAGGATCGTTACGACTAGCATCGATG SEQ ID No. 138 >PB.97.126.C_44-G6
GGGAGAGGAGAGAACGTTCTCGGCTTGGAAAAATAGCTTTTGGG- CATCC
GGGATCGTTACGACTAGCATCGATG SEQ ID No. 139 >PB.97.126.C_44-H6
GGGAGAGGAGAGAACGTTCTCGGGTTCAAGGAACATGATAGCTA- GAACC
CGCGGATCGTTACGACTAGCATCGATG SEQ ID No. 140 >PB.97.126.C_44-A7
GGGAGAGGAGAGAACGTTCTCGGGTTCAAGGAACATGATAGTTA- GAACC
CGCGGATCGTTACGACTAGCATCGATG SEQ ID No. 141 >PB.97.126.C_44-B7
GGGAGAGGAGAGAACGTTCTCGTGGGCAGGGAACACAATAGTTA- GCCTA
CGCGGATCGTTACGACTAGCATCGATG SEQ ID No. 142 >PB.97.126.C_44-C7
GGGAGAGGAGAGAACGTTCTCGCGTGAAAGGAACACAATAGTTA- TCGTG
CGGGATCGTTACGACTAGCATCGATG SEQ ID No. 143 >PB.97.126.C_44-D7
GGGAGAGGAGAGAACGTTCTCGCGAGGTTTATCCTAGACGACTA- ACCGC
CTGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 144 >PB.97.126.C_44-F7
GGGAGAGGAGAGAACGTTCTCGTCTGCTAGGAACACAATAGTTA- GCATT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 145 >PB.97.126.C_44-G7
GGGAGAGGAGAGAACGTTCTCGCACAAGGAACTACGAGTTAGTG- TGGGA
GTGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 146 >PB.97.126.C_44-H7
GGGAGAGGAGAGAACGTTCTCGTGACACGAGGAACTTAGAGTTA- GTAGC
ACGAGGATCGTTACGACTAGCATCGATG SEQ ID No. 147 >PB.97.126.C_44-A8
GGGAGAGGAGAGAACGTTCTCGGCGGCGAAGGAACACAATAGTT- ACGTC
CCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 148 >PB.97.126.C_44-B8
GGGAGAGGAGAGAACGTTCTCGAGCCCAAAAAAGCTGAAGTACT- TTGGG
CAGGGATCGTTACGACTAGCATCGATG
[0212]
14TABLE 9 Corresponding cDNAs of the Thrombin Aptamer Sequences -
2'-OMe AUGC (r/mGmH, each G has a 90% probability of having a
2'-OMe group incorporated therein) SEQ ID No. 149
>PB.97.126.D_44-D8
GGGAGAGGAGAGAACGTTCTCGGTACAAGGAACACAATAGTTAGTGCCG
TGGGATCGTTACGACTAGCATCGATG SEQ ID No. 150 >PB.97.126.D_44-E8
GGGAGAGGAGAGAACGTTCTCGGATCGTTACGACTAGCATCGAT- G SEQ ID No. 151
>PB.97.126.D_44-G8
GGGAGAGGAGAGAACGTTCTCGTGCGCAAGGAACACAATAGTTAGGGCG
CGAGGATCGTTACGACTAGCATTGATG SEQ ID No. 152 >PB.97.126.D_44-H8
GGGAGAGGAGAGAACGTTCTCGGAATGGAAGGAACACAATAGTT- ACCAG
ACGGGATCGTTACGACTAGCATCGATG SEQ ID No. 153 >PB.97.126.D_44-A9
GGGAGAGGAGAGAACGTTCTCGTCTGCAAGGAACACAATAGTTA- GCATT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 154 >PB.97.126.D_44-B9
GGGAGAGGAGAGAACGTTCTCGAGACAAGACAGCTGGAGGACTA- AGTCA
CGAGGATCGTTACGACTAGCATCGATG SEQ ID No. 155 >PB.97.126.D_44-C9
GGGAGAGGAGAGAACGTTCTCGATGCCCGCAAAGGAACACGATA- GTTAT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 156 >PB.97.126.D_44-D9
GGGAGAGGAGAGAACGTTCTCGTCTGNNAGGAACACAATATTTA- GCATT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 157 >PB.97.126.D_44-E9
GGGAGAGGAGAGAACGTTCTCGAATGTGCGGAGCAGTATTGGTA- CACTT
TCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 158 >PB.97.126.D_44-F9
GGGAGAGGAGAGAACGTTCTCGCCAAGGAACACAATAGTTAGGT- GAGAA
TCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 159 >PB.97.126.D_44-G9
GGGAGAGGAGAGAACGTTCTCGCCAAGGAACACAATAGTTAGGT- GAGAA
TCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 160 >PB.97.126.D_44-H9
GGGAGAGGAGAGAACGTTCTCGGGAAGCAAGGAACTTAGAGTTA- GTTGA
CCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 161 >PB.97.126.D_44-A10
GGGAGAGGAGAGAACGTTCTCGTGGGCAAGGAACACAATAGTT- AGCCTA
CGCGGATCGTTACGACTAGCATCGATG SEQ ID No. 162 >PB.97.126.D_44-B10
GGGAGAGGAGAGAACGTTCTCGTCGGGCATGGAACACAATAGT- TAGACC
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 163 >PB.97.126.D_44-C10
GGGAGAGGAGAGAACGTTCTCGGTCGCAAGGAACATAATAGTT- AGCGGA
GGGGATCGTTACGACTAGCATCGATG SEQ ID No. 164 >PB.97.126.D_44-D10
GGGAGAGGAGAGAACGTTCTCGTCTGCAAGGAACACAATAGTT- AGCATT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 165 >PB.97.126.D_44-E10
GGGAGAGGAGAGAACGTTCTCGCCGACAATCAGCTCGGATCGT- GTGCTA
CGCTGGATCGTTACGACTAGCATCGATG
[0213]
15TABLE 10 Corresponding cDNAs of the Thrombin Aptamer Sequences -
alternately "r/mGmH" and 2'-OMe AUC, 2'-F G (toggle). SEQ ID No.
166 >PB.97.126.E_44-F10
GGGAGAGGAGAGAACGTTCTCGAGACAAGATAGCTGAAGGAC- TAAGTCA
CGAGGGATCGTTACGACTAGCATCGATG SEQ ID No. 167 >PB.97.126.E_44-G10
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGAAGGA- CTAAGTTT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 168 >PB.97.126.E_44-H10
GGGAGAGGAGAGAACGTTCTCGGAGNCAAGGAAACNAATAT- TTAGGCTC
ANTGGNNNCNTTNCANCTAGCNNCNNTA SEQ ID No. 169 >PB.97.126.E_44-A11
GGGAGAGGAGAGAACGTTCTCGTCTGCAAGGAACACA- ATAGTTAGCATT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 170 >PB.97.126.E_44-B11
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGA- AGGACTAAGTTT
ACGGGATCGTTACGACTAGCATCGATG SEQ ID No. 171 >PB.97.126.E_44-C11
GGGAGAGGAGAGAACGTTCTCGGATCGTTACGACTAG- CATCGATG SEQ ID No. 172
>PB.97.126.E_44-D11
GGGAGAGGAGAGAACGTTCTCGGTGATAGTACTCACATAGAAATGGCTA
CACTGGGATCGTTACGACTAGCATCGATG SEQ ID No. 173 >PB.97.126.E_44-E11
GGGAGAGGAGAGAACGTTCTCGCCTGGGCAAGGAACAGAAAAG- TTAGCG
CCAGGATCGTTACGACTAGCATCGATG SEQ ID No. 174 >PB.97.126.E_44-F11
GGGAGAGGAGAGAACGTTCTCGTAACGGACAAAAGGAACCGGG- AAGTTA
TCTGGATCGTTACGACTAGCATCGATG SEQ ID No. 175 >PB.97.126.E_44-G11
GGGAGAGGAGAGAACGTTCTCGCGCACAAGATAGAGAAGACTA- AGTCCG
CGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 176 >PB.97.126.E_44-H11
GGGAGAGGAGAGAACGTTCTCGCGCACAAGATAGAGAAGACTA- AGTTCG
CGGGGATCGTTACGACTAGCATCGATG SEQ ID No. 177 >PB.97.126.E_44-A12
GGGAGAGGAGAGAACGTTCTCGCGCCAATAAAGCTGGAGTACT- TAGAGC
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 178 >PB.97.126.E_44-B12
GGGAGAGGAGAGAACGTTCTCGGGAAACAAGGAACTTAGAGTT- AGTTGA
CCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 179 >PB.97.126.E_44-C12
GGGAGAGGAGAGAACGTTCTCGCTAGCAAGATAGGTGGGACTA- AGCTAG
TGAGGATCGTTACGACTAGCATCGATG SEQ ID No. 180 >PB.97.126.E_44-D12
GGGAGAGGAGAGAACGTTCTCGTCGAAGGGGAGCTTTGTCTCG- GGACAG
AACGGATCGTTACGACTAGCATCGATG SEQ ID No. 181 >PB.97.126.E_44-E12
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGAAGGACT- AAGTTT
ACGGGATCGTTACGACTAGCATCGATG SEQ ID No. 182 >PB.97.126.E_44-G12
GGGAGAGGAGAGAACGTTCTCGGAACAAGATAGCTGAAGGACT- AAGTTT
GCGGGATCGTTACGACTAGCATCGATG SEQ ID No. 183 >PB.97.126.E_44-H12
GGGAGAGGAGANNTCCCCNCNCGGAAAAANAAAAAAGAAGAAN- TANGTT
NGGGGGATCGTTACGACTAGCATCGATG
[0214]
16TABLE 11 Stabilized Aptamer Sequences (each G residue has 90%
probability of being substituted with a 2'-OMe group, "3T" refers
to an inverted thymidine nucleotide attached to the phosphodiester
backbone at the 5' position, the resulting oligo having two 5'-OH
ends and is thus resistant to 3' nucleases). SEQ ID No. 184 ARC224
- Stabilized VEGF Aptamer
5'mCmGmAmUmAmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCmGmCm GmCmAmUmUmCmG-3T
SEQ ID No. 185 ARC225 - Stabilized VEGF Aptamer
5'mCmGmAmUmAmUGmCmAGmUmUmUGmAGmAmAGmUmCGmCGmCmAmUm UmCmG-3T SEQ ID
No. 186 ARC226 Single-hydroxy VEGF aptamer
5'mGmAmUmCmAmUmGmCmAmUGmUmGmGmAmUmCmGmCmGmGmAmUmC- 3T SEQ ID No.
187 ARC245 VEGF Aptamer
5'mAmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCmGmCmGmCmAmU- 3T SEQ ID No. 188
ARC259 hVEGF Aptamer - C-G base pair swap of ARC245 (2nd base pair
in) which has improved binding over ARC245.
5'mAmCmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCmGmCmGmCmGmU- 3'
Example 2
2'-OMe SELEX.TM.
[0215] Libraries of transcription templates were used to generate
pools of RNA oligonucleotides incorporating 2'-O-methyl NTPs under
various transcription conditions. The transcription template
(ARC256) and the transcription conditions are described below as
rRmY (SEQ ID NO:456), rGmH (SEQ ID NO:462), r/mGmH (SEQ ID NO:463),
and dRmY (SEQ ID NO:464). The unmodified RNA transcript is
represented by SEQ ID NO:468.
17 ARC256: DNA transcription template
5'-CATCGATCGATCGATCGACAGCGNNNNNNNN (SEQ ID NO:453)
NNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCT
CTCCTCTCCCTATAGTGAGTCGTATTA-3'
[0216] The ARC256 RNA transcription product is:
18 5'-GGGAGAGGAGAGAACGUUCUACNNNNNNNNN (SEQ ID NO:468)
NNNNNNNNNNNNNNNNNNNNNCGCUGUCGAUCGA UCGAUCGAUG-3'
[0217] The transcription conditions were varied as follows where
1.times.Tc buffer is 200 mM HEPES, 40 mM DTT, 2 mM Spermidine,
0.01% Triton X-100, pH 7.5.
[0218] When 2'-OMe C and U and 2'-OH A and G (rRmY) conditions were
used, the transcription reaction conditions were 1.times.Tc buffer,
50-200 nM double stranded template (200 nm template was used for
round 1, and for subsequent rounds approximately 50 nM, a {fraction
(1/10)} dilution of an optimized PCR reaction, using conditions
described herein, was used), 9.6 mM MgCl.sub.2, 2.9 mM MnCl.sub.2,
2 mM each base, 10% PEG-8000, 0.25 units inorganic pyrophosphatase,
and 1.5 units Y639F/H784A T7 RNA polymerase. One unit of the
Y639F/H784A mutant T7 RNA polymerase is defined as the amount of
enzyme required to incorporate 1 nmole of 2'-OMe NTPs into
transcripts under the r/mGmH conditions. One unit of inorganic
pyrophosphatase is defined as the amount of enzyme that will
liberate 1.0 mole of inorganic orthophosphate per minute at pH 7.2
and 25.degree. C.
[0219] When 2'-OMe A, C, and U and 2'-OH G (rGmH) conditions were
used, the transcription reaction conditions were 1.times.Tc buffer,
50-200 nM double stranded DNA template (200 nm template was used
for round 1, and for subsequent rounds approximately 50 nM, a
{fraction (1/10)} dilution of an optimized PCR reaction, using
conditions described herein was used), 9.6 mM MgCl.sub.2, 2.9 mM
MnCl.sub.2, 2 mM each base, 10% PEG-8000, 0.25 units inorganic
pyrophosphatase, and 1.5 units Y639F single mutant T7 RNA
polymerase. One unit of the Y639F mutant T7 RNA polymerase is
defined as the amount of enzyme required to incorporate 1 nmole of
2'-OMe NTPs into transcripts under the r/mGmH conditions.
[0220] When all 2'-OMe nucleotides (r/mGmH) conditions were used,
the reaction conditions were 1.times.Tc buffer, 50-200 nM double
stranded template (200 nm template was used for round 1, and for
subsequent rounds approximately 50 nM, a {fraction (1/10)} dilution
of an optimized PCR reaction, using conditions described herein was
used), 6.5 mM MgCl.sub.2, 2 mM MnCl.sub.2, 1 mM each base, 30 .mu.M
GTP, 1 mM GMP, 10% PEG-8000, 0.25 units inorganic pyrophosphatase,
and 1.5 units Y639F/H784A T7 RNA polymerase.
[0221] When deoxy purines, A and G, and 2'-OMe pyrimidines (dRmY)
conditions were used, the reaction conditions were 1.times.Tc
buffer, 50-300 nM double stranded template (300 nm template was
used for round 1, and for subsequent rounds approximately 50 nM, a
{fraction (1/10)} dilution of an optimized PCR reaction, using
conditions described herein was used), 9.6 mM MgCl.sub.2, 2.9 mM
MnCl.sub.2, 2 mM each base, 30 .mu.M GTP, 2 mM Spermine, 10%
PEG-8000, 0.25 units inorganic pyrophosphatase, and 1.5 units Y639F
single mutant RNA polymerase.
[0222] These pools were then used in SELEX.TM. to select for
aptamers against the following targets: IgE, IL-23, PDGF-BB,
thrombin and VEGF. A plot of dRmY Round 6, 7, 8, and unselected
sequences binding to target IL-23 is shown in FIG. 14, and a plot
of dRmY Round 6, 7, and unselected sequences binding to target
PDGF-BB is shown in FIG. 14.
Example 3
dRmY SELEX.TM. of Aptamers Against IgE
[0223] While fully 2'-OMe substituted oligonucleotides are the most
stable modified aptamers, substituting the purines with deoxy
purine nucleotides also results in stable transcripts. When dRmY
(deoxy purines, A and G, and 2'-OMe pyrimidines) transcription
conditions are used, the products are very DNase-resistant and
useful as stable therapeutics. This result is surprising since the
composition of the dRmY transcripts is approximately 50% DNA, which
is notoriously easily degraded by nucleases. Also, when dRmY
transcription conditions are used, there is no requirement for a
2'-OH GTP spike. Studies have shown that approximately the same
amount of dRmY transcripts having modified nucleotides are produced
with 2'-OH GTP doping as without 2'-OH GTP doping. Accordingly,
under dRmY transcription conditions, 2'-OH GTP doping is optional.
Libraries of transcription templates were used to generate pools of
oligonucleotides incorporating 2'-O-methylpyrimidine NTPs (U and C)
and deoxy purines (A and G) NTPs under various transcription
conditions. The transcription template (ARC256) and the
transcription conditions are described below as dRmY.
19 ARC256: DNA transcription template
5'-CATCGATCGATCGATCGACAGCGNNNNNNNN (SEQ ID NO:453)
NNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCT
CTCCTCTCCCTATAGTGAGTCGTATTA-3'
[0224] The ARC256 dRmY RNA transcription product is:
20 5'-GGGAGAGGAGAGAACGUUCUACNNNNNNNNN (SEQ ID NO:464)
NNNNNNNNNNNNNNNNNNNNNCGCUGUCGAUCGA UCGAUCGAUG-3'
[0225] When deoxy purines, A and G, and 2'-OMe pyrimidines (dRmY)
conditions were used, the reaction conditions were 1.times.Tc
buffer, 50-300 nM double stranded template (300 nm template was
used for round 1, and for subsequent rounds approximately 50 nM, a
{fraction (1/10)} dilution of an optimized PCR reaction, using
conditions described herein, was used), 9.6 mM MgCl.sub.2, 2.9 mM
MnCl.sub.2, 2 mM each base, 30 .mu.M GTP, 2 mM Spermine, 10%
PEG-8000, 0.25 units inorganic pyrophosphatase, and 1.5 units Y639F
single mutant RNA polymerase.
[0226] These pools were then used in SELEX.TM. to select for
aptamers against IgE as a target. The sequences obtained after
round 6 of SELEX.TM. as described above are listed in Table 12
below. A plot of Round 6 sequences bound with increasing target IgE
concentration is shown in FIG. 8.
21TABLE 12 Corresponding cDNAs of the Round 6 sequences of dRmY
SELEX .TM. against IgE. SEQ ID No.190 IgE A5
GGGAGAGGAGAGAACGTTCTACAGGCAGTTCTGGGGACCCAT- GGGGGAAG
TGCGCTGTCGATCGATCGATCGATG SEQ ID No.191 IgE A6
GGGAGAGGAGAGAACGTTCTACGATTAGCAGGGAGGGAGAGTGCGAAGAG
GACGCTGTCGATCGATCGATCGATG SEQ ID No.192 IgE A7
GGGAGAGGAGAGAACGTTCTACACTCTGGGGACCCGTGGGGGAGTGCAG
CAACGCTGTCGATCGATCGATCGATG SEQ ID No.193 IgE A8
GGGAGAGGAGAGAACGTTCTACAAGCAGTTCTGGGGACCCATGGGGGAA
GTGCGCTGTCGATCGATCGATCGATG SEQ ID No.194 IgE B5
GGGAGAGGAGAGAACGTTCTACGAGGTGAGGGTCTACAATGGAGGGATG
GTCGCTGTCGATCGATCGATCGATG SEQ ID No.195 IgE B6
GGGAGAGGAGAGAACGTTCTACCCGCAGCATAGCCTGNGGACCCATGNG
GGGCGCTGTCGATCGATCGATCGATG SEQ ID No.196 IgE B7
GGGAGAGGAGAGAACGTTCTACTGGGGGGCGTGTTCATTAGCAGCGTCG
TGTCGCTGTCGATCGATCGATCGATG SEQ ID No.197 IgE B8
GGGAGAGGAGAGAACGTTCTACAGGCAGTTCTGGGGACCCATGGGGGAA
GTGCGCTGTCGATCGATCGATCGATG SEQ ID No.198 IgE C5
GGGAGAGGAGAGAACGTTCTACGCAGCGCATCTGGGGACCCAAGAGGGG
ATTCGCTGTCGATCGATCGATCGATG SEQ ID No.199 IgE C6
GGGAGAGGAGAGAACGTTCTACAGGCAGTTCTGGGGACCCATGGGGGAA
GTGCGCTGTCGATCGATCGATCGATG SEQ ID No.200 IgE C7
GGGAGAGGAGAGAACGTTCTACGGGATGGGTAGTTGGATGGAAATGGGA
ACGCTGTCGATCGATCGATCGATG SEQ ID No.201 IgE C8
GGGAGAGGAGAGAACGTTCTACGAGGTGTAGGGATAGAGGGGTGTAGGT
AACGCTGTCGATCGATCGATCGATG SEQ ID No.202 IgE D5
GGGAGAGGAGAGAACGTTCTACAGGAGTGGAGCTACAGAGAGGGTTAGG
GGTCGCTGTCGATCGATCGATCGATG SEQ ID No.203 IgE D6
GGGAGAGGAGAGAACGTTCTACGGATGTTGGGAGTGATAGAAGGAAGGG
GAGCGCTGTCGATCGATCGATCGATG SEQ ID No.204 IgE D7
GGGAGAGGAGAGAACGTTCTACAGGCAGTTCTGGGGACCCATGGGGGAA
GTGCGCTGTCGATCGATCGATCGATG SEQ ID No.205 IgE D8
GGGAGAGGAGAGAACGTTCTACAGGCAGTTCTGGGGACCCATGGGGGAA
GTGCGCTGTCGATCGATCGATCGATG SEQ ID No.206 IgE E5
GGGAGAGGAGAGAACGTTCTACAGGCAGTTCTGGGGACCCATGGGGGAA
GTGCGCTGTCGATCGATCGATCGATG SEQ ID No.207 IgE E6
GGGAGAGGAGAGAACGTTCTACTTGGGGTGGAAGGAGTAAGGGAGGTGC
TGATCGCTGTCGATCGATCGATCGATG SEQ ID No.208 IgE E7
GGGAGAGGAGAGAACGTTCTACGTATTAGGGGGGAAGGGGAGGAATAGA
TCACGCTGTCGATCGATCGATCGATG SEQ ID No.209 IgE E8
GGGAGAGGAGAGAACGTTCTACAGGGAGAGAGTGTTGAGTGAAGAGGAG
GAGTCGCTGTCGATCGATCGATCGATG SEQ ID No.210 IgE F5
GGGAGAGGAGAGAACGTTCTACATTGTGCTCCTGGGGCCCAGTGGGGAG
CCACGCTGTCGATCGATCGATCGATG SEQ ID No.211 IgE F6
GGGAGAGGAGAGAACGTTCTACGAGCAGCCCTGGGGCCCGGAGGGGGAT
GGTCGCTGTCGATCGATCGATCGATG SEQ ID No.212 IgE F7
GGGAGAGGAGAGAACGTTCTACAGGCAGTTCTGGGGACCCATGGGGGAA
GTGCGCTGTCGATCGATCGATCGATG SEQ ID No.213 IgE F8
GGGAGAGGAGAGAACGTTCTACCAACGGCATCCTGGGCCCCACAGGGGA
TGTCGCTGTCGATCGATCGATCGATG SEQ ID No.214 IgE G5
GGGAGAGGAGAGAACGTTCTACGAGTGGATAGGGAAGAAGGGGAGTAGT
CACGCTGTCGATCGATCGATCGATG SEQ ID No.215 IgE G6
GGGAGAGGAGAGAACGTTCTACCCGCAGCATAGCCTGGGGACCCATGGG
GGGCGCTGTCGATCGATCGATCGATG SEQ ID No.216 IgE G7
GGGAGAGGAGAGAACGTTCTACGGTCGCGTGTGGGGGACGGATGGGTAT
TGGTCGCTGTCNATCGATCGATCNATG SEQ ID No.217 IgE G8
GGGAGAGGAGAGAACGTTCTACCCGCAGCATAGCCTGGGGACCCATGGG
GGGCGCTGTCGATCGATCGATCGATG SEQ ID No.218 IgE H5
GGGAGAGGAGAGAACGTTCTACCCGCAGCATAGCCTGGGGACCCATGGG
GGGCGCTGTCGATCGATCGATCGATG SEQ ID No.219 IgE H6
GGGAGAGGAGAGAACGTTCTACGGGGTTACGTCGCACGATACATGCATT
CATCGCTGTCGATCGATCGATCGATG SEQ ID No.220 IgE H7
GGGAGAGGAGAGAACGTTCTACTAGCGAGGAGGGGTTTTCTATTTTTGC
GATCGCTGTCGATCGATCGATCGATG
Example 4
dRmY SELEX.TM. of Aptamers Against Thrombin
[0227] While fully 2'-OMe substituted oligonucleotides are the most
stable modified aptamers, substituting the purines with deoxy
purine nucleotides also results in stable transcripts. When dRmY
(deoxy purines, A and G, and 2'-OMe pyrimidines) transcription
conditions are used, the products are very DNase-resistant and
useful as stable therapeutics. This result is surprising since the
composition of the dRmY transcripts is approximately 50% DNA, which
is notoriously easily degraded by nucleases. Also, when dRmY
transcription conditions are used, there is no requirement for a
2'-OH GTP spike. Libraries of transcription templates were used to
generate pools of oligonucleotides incorporating
2'-O-methylpyrimidine NTPs (U and C) and deoxy purines (A and G)
NTPs under various transcription conditions. The transcription
template (ARC256) and the transcription conditions are described
below as dRmY.
22 ARC256: DNA transcription template
5'-dCATCGATCGATCGATCGACAGCGNNNNNNN (SEQ ID NO:453)
NNNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTC
TCTCCTCTCCCTATAGTGAGTCGTATTA-3'
[0228] The ARC256 dRmY RNA transcription product is:
23 5'-GGGAGAGGAGAGAACGUUCUACNNNNNNNNN (SEQ ID NO:464)
NNNNNNNNNNNNNNNNNNNNNCGCUGUCGAUCGA UCGAUCGAUG-3'
[0229] When deoxy purines, A and G, and 2'-OMe pyrimidines (dRmY)
conditions were used, the reaction conditions were 1.times.Tc
buffer, 50-300 nM double stranded template (300 nm template was
used for round 1, and for subsequent rounds a {fraction (1/10)}
dilution of an optimized PCR reaction, using conditions described
herein, was used), 9.6 mM MgCl.sub.2, 2.9 mM MnCl.sub.2, 2 mM each
base, 30 .mu.M GTP, 2 mM Spermine, 10% PEG-8000, 0.25 units
inorganic pyrophosphatase, and 1.5 units Y639F single mutant RNA
polymerase.
[0230] These pools were then used in SELEX.TM. to select for
aptamers against thrombin as a target. The sequences obtained after
round 6 of SELEX.TM. as described above are listed in Table 13
below. A plot of Round 6 sequences bound to target thrombin is
shown in FIG. 9.
24TABLE 13 Corresponding cDNAs of the Round 6 sequences of dRmY
SELEX .TM. against thrombin. SEQ ID No.221 Thrombin A1
GGGAGAGGAGAGAACGTTCTACGTGTGATGGGG- TGAGAGGATGAGTTAGT
GACGCTGTCGATCGATCGATCGATG SEQ ID No.222 Thrombin A2
GGGAGAGGAGAGAACGTTCTACAATGGGAGGGTAATAGTGATGAG- GAGAG
GCGCTGTCGATCGATCGATCGATG SEQ ID No.223 Thrombin A3
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.224 Thrombin A4
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.225 Thrombin B1
GGGAGAGGAGAGAACGTTCTACAGGTAGCGTGAGGGGGTGTTAATAGAGG
GGCGCTGTCGATCGATCGATCGATG SEQ ID No.226 Thrombin B2
GGGAGAGGAGAGAACGTTCTACGATAGGATGGGTGGGACAGGAGAGGGAG
TGCGCTGTCGATCGATCGATCGATG SEQ ID No.227 Thrornbin B3
GGGAGAGGAGAGAACGTTCTACCAGTGAGGGCAGTGTCAGATTGAGAGGA
GGCGCTGTCGATCGATCGATCGATG SEQ ID No.228 Thrombin B4
GGGAGAGGAGAGAACGTTCTACCTTGCCTAACAGGAGGTGGAGTATTGGA
CCCGCTGTCGATCGATCGATCGATG SEQ ID No.229 Thrombin C1
GGGAGAGGAGAGAACGTTCTACCTTGCCTAACAGGAGGTGGAGTATTGGA
CCCGCTGTCGATCGATCGATCGATG SEQ ID No.230 Thrombin C2
GGGAGAGGAGAGAACGTTCTACGTCGTGAGTAATGGCTCGTAGATGAGGT
CGCTGTCGATCGATCGATCGATG SEQ ID No.231 Throinbin C3
GGGAGAGGAGAGAACGTTCTACGGGATTAAGAGGGGAGAGGAGCAGTTGA
GCGCTGTCGATCGATCGATCGATG SEQ ID No.232 Thrombin C4
GGGAGAGGAGAGAACGTTCTACTCCGGTTGGGGTATCAGGTCTACGGACT
GACGCTGTCGATCGATCGATCGATG SEQ ID No.233 Thrombin D1
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.234 Thrombin D2
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.235 Thrombin D3
GGGAGAGGAGAGAACGTTCTACATGACAAGAGGGGGTTGTGTGGGATGGC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.236 Thrombin D4
GGGAGAGGAGAGAACGTTCTACACAGGGAGGGGAGCGGAGAGGAGAGAGG
GTACGCTGTCGATCGATCGATCGATG SEQ ID No.237 Thrombin E1
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.238 Thrombin E2
GGGAGAGGAGAGAACGTTCTACGTCGTGAGTAATGGCTCGTAGATGAGGT
CGCTGTCGATCGATCGATCGATG SEQ ID No.239 Thrombin E4
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.240 Thrombin F1
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.241 Thrombin F2
GGGAGAGGAGAGAACGTTCTACCTTGCCTAACAGGAGGTGGAGTATTGGA
CCCGCTGTCGATCGATCGATCGATG SEQ ID No.242 Thrombin F3
GGGAGAGGAGAGAACGTTCTACGGCTATGCGTCGTGAGTCAATGGCCCGC
ATCGCTGTCGATCGATCGATCGATG SEQ ID No.243 Thrombin F4
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAGTGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.244 Thrombin G1
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.245 Thrombin G2
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.246 Thrombin G3
GGGAGAGGAGAGAACGTTCTACCTTGTCTAACAGGAGGTGGAGTATTGGA
CCCGCTGTCGATCGATCGATCGATG SEQ ID No.247 Thrombin G4
GGGAGAGGAGAGAACGTTCTACGACTTTGAGGGTGGTGAGAGTGGAAGAG
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.248 Thrombin H1
GGGAGAGGAGAGAACGTTCTACGGTAGGGTATGACCAGGGAGGTATTGGA
GGCGCTGTCGATCGATCGATCGATG SEQ ID No.249 Thrombin H2
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.250 Thrombin H3
GGGAGAGGAGAGAACGTTCTACGGGTCGTGAGATAATGGCTCCCGTATTC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.251 Thrombin H4
GGGAGAGGAGAGAACGTTCTACGTTATGCATGTGGAGAGTGAGAGAGGGC
GCTGTCGATCGATCGATCGATG
Example 5
dRmY SELEX.TM. of Aptamers Against VEGF
[0231] While fully 2'-OMe substituted oligonucleotides are the most
stable modified aptamers, substituting the purines with deoxy
purine nucleotides also results in stable transcripts. When dRmY
(deoxy purines, A and G, and 2'-OMe pyrimidines) transcription
conditions are used, the products are very DNase-resistant and
useful as stable therapeutics. This result is surprising since the
composition of the dRmY transcripts is approximately 50% DNA RNA,
which is notoriously easily degraded by nucleases. Also, when dRmY
transcription conditions are used, there is no requirement for a
2'-OH GTP spike. Libraries of transcription templates were used to
generate pools of oligonucleotides incorporating
2'-O-methylpyrimidine NTPs (U and C) and deoxy purines (A and G)
NTPs under various transcription conditions. The transcription
template (ARC256) and the transcription conditions are described
below as dRmY.
25 ARC2S6: DNA transcription template
5'-CATCGATCGATCGATCGACAGCGNNNNNNNN (SEQ ID NO:453)
NNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCT
CTCCTCTCCCTATAGTGAGTCGTATTA-3'
[0232] ARC256 dRmY transcription product is:
26 5'-GGGAGAGGAGAGAACGUUCUACNNNNNNNNN (SEQ ID NO:464)
NNNNNNNNNNNNNNNNNNNNNCGCUGUCGAUCGA UCGAUCGAUG-3'
[0233] When deoxy purines, A and G, and 2'-OMe pyrimidines (dRmY)
conditions were used, the reaction conditions were 1.times.Tc
buffer, 50-300 nM double stranded template (300 nm template was
used for round 1, and for subsequent rounds a {fraction (1/10)}
dilution of an optimized PCR reaction, using conditions described
herein, was used), 9.6 mM MgCl.sub.2, 2.9 mM MnCl.sub.2, 2 mM each
base, 30 .mu.M GTP, 2 mM Spermine, 10% PEG-8000, 0.25 units
inorganic pyrophosphatase, and 1.5 units Y639F single mutant RNA
polymerase.
[0234] These pools were then used in SELEX.TM. to select for
aptamers against VEGF as a target. The sequences obtained after
round 6 of SELEX.TM. as described above are listed in an alignment
show in Table 14 below. A plot of Round 6 sequences bound to target
VEGF is shown in FIG. 10.
27TABLE 14 Corresponding cDNAs of the Round 6 sequences of dRmY
SELEX .TM. against VEGF. SEQ ID No.252 VEGF A9
GGGAGAGGAGAGAACGTTCTACCATGTCTGCGGGAGGTGAG- TAGTGATCC
TGCGCTGTCGATCGATCGATCGATG SEQ ID No.253 VEGF A10
GGGAGAGGAGAGAACGTTCTACAGAGTGGGAGGGATGTGTGACACAGGTA
GGCGCTGTCGATCGATCGATCGATG SEQ ID No.254 VEGF A11
GGGAGAGGAGAGAACGTTCTACGCTCCATGACAGTGAGGTGAGTAGTGAT
CGCTGTCGATCGATCGATCGATG SEQ ID No.255 VEGF A12 GGGAGAGGAGAGAACGTTCT
CGATGCTGACAGGGTGTGTTCAGTAATGG CTCGCTGTCGATCGATCGATCGATG SEQ ID
No.256 VEGF B9 GGGAGAGGAGAGAACGTTCTACCAGCAAACAGGGTCAGGTGAGTAGTGAT
GACGCTGTCGATCGATCGATCGATG SEQ ID No.257 VEGF B10
GGGAGAGGAGAGAACGTTCTACGACAAGCCGGGGGTGTTCAGTAGTGGCA
ACCGCTGTCGATCGATCGATCGATG SEQ ID No.258 VEGF B11
GGGAGAGGAGAGAACGTTCTACATATGGCGCTGGAGGTGAGTAATGATCG
TGCGCTGTCGATCGATCGATCGATG SEQ ID No.259 VEGF B12
GGGAGAGGAGAGAACGTTCTACGGGGCGATAGCGTTCAGTAGTGGCGCCG
GTCGCTGTCGATCGATCGATCGATG SEQ ID No.260 VEGF C9
GGGAGAGGAGAGAACGTTCTACATAGCGGACTGGGTGCATGGAGCGGCGC
ACGCTGTCGATCGATCGATCGATG SEQ ID No.261 VEGF C10
GGGAGAGGAGAGAACGTTCTACGGGTCAACAGGGGCGTTCAGTAGTGGCG
GCGCTGTCGATCGATCGATCGATG SEQ ID No.262 VEGF C11
GGGAGAGGAGAGAACGTTCTACGCATGCGAGCTGAGGTGAGTAGTGATCA
GTCGCTGTCGATCGATCGATCGATG SEQ ID No.263 VEGF C12
GGGAGAGGAGAGAACGTTCTACATGCGACAGGGGAGTGTTCAGTAGTGGC
ACGCTGTCGATCGATCGATCGATG SEQ ID No.264 VEGF D9
GGGAGAGGAGAGAACGTTCTACCCCATCGTATGGAGTGCGGAACGGGGCA
TACGCTGTCGATCGATCGATCGATG SEQ ID No.265 VEGF D10
GGGAGAGGAGAGAACGTTCTACAGTGAGGCGGGAGCGTTTCAGTAATGGC
GCTGTCGATCGATCGATCGATG SEQ ID No.266 VEGF D12
GGGAGAGGAGAGAACGTTCTACACAGCGTCGGGTGTTCAGTAATGGCGCA
GCGCTGTCGATCGATCGATCGATG SEQ ID No.267 VEGF E9
GGGAGAGGAGAGAACGTTCTACGGTGTTCAGTAGTGGCACAGGAGGAAGG
GATGCTGTCGATCGATCGATCGATG SEQ ID No.268 VEGF E10
GGGAGAGGAGAGAACGTTCTACAGTTCAGGCGTTAGGCATGGGTGTCGCT
TTCGCTGTCGATCGATCGATCGATG SEQ ID No.269 VEGF E11
GGGAGAGGAGAGAACGTTCTACATGCGACATGCGAGTGTTCAGTAGCGGC
AGCGCTGTCGATCGATCGATCGATG SEQ ID No.270 VEGF E12
GGGAGAGGAGAGAACGTTCTACCTATGGCGTTACAGCGAGGTGAGTAGTG
ATCGCTGTCGATCGATCGATCGATG SEQ ID No.271 VEGF F9
GGGAGAGGAGAGAACGTTCTACCAGCCGATCCAGCCAGGCGTTCAGTAGT
GGCGCTGTCGATCGATCGATCGATG SEQ ID No.272 VEGF F10
GGGAGAGGAGAGAACGTTCTACGGCACAGGCACGGCGAGGTGAGTAATGA
TCGCTGTCGATCGATCGATCGATG SEQ ID No.273 VEGF G9
GGGAGAGGAGAGAACGTTCTACTGTGGACAGCGGGAGTGCGGAACGGGGT
CGCTGTCGATCGATCGATCGATG SEQ ID No.274 VEGF G10
GGGAGAGGAGAGAACGTTCTACTGATGCTGCGAGTGCATGGGGCAGGCGC
TTCGCTGTCGATCGATCGATCGATG SEQ ID No.275 VEGF G11
GGGAGAGGAGAGAACGTTCTACGGTACAATGGGAATGACAGTGATGGGTA
GCCGCTGTCGATCGATCGATCGATG SEQ ID No.276 VEGF G12
GGGAGAGGAGAGAACGTTCTACATGGACAGCGAAGCATGGGGGAGGCGCA
CGCTGTCGATCGATCGATCGATG SEQ ID No.277 VEGF H9
GGGAGAGGAGAGAACGTTCTACTGGGAGCGACAGTGAGCATGGGGTAGGC
GCCGCTGTCGATCGATCGATCGATG SEQ ID No.278 VEGF H11
GGGAGAGGAGAGAACGTTCTACCGGCGAGCAGGTGTTCAGTAGTGGCTTT
GCGCTGTCGATCGATCGATCGATG SEQ ID No.279 VEGF H12
GGGAGAGGAGAGAACGTTCTACGATCAGTGAGGGAGTGCAGTAGTGGCTC
GTCGCTGTCGATCGATCGATCGATG
Example 6
Plasma Stability of 2'-OMe NTPs (mN) and dRmY Oligonucleotides
[0235] An oligonucleotide of two sequences linked by a polyethylene
glycol polymer (PEG) was synthesized in two versions: (1) with all
2'-OMe NTPs (mN): 5'-GGAGCAGCACC-3' (SEQ ID
NO:457)-[PEG]-GGUGCCAAGUCGUUGCUCC-3' (SEQ ID NO:458) and (2) with
2'-OH purine NTPs and 2'-OMe pyrimidines (dRmY) GGAGCAGCACC-3' (SEQ
ID NO:465)-[PEG]-GGUGCCAAGUCGUUGCUCC-3' (SEQ ID NO:466). These
oligonucleotides were evaluated for full length stability. FIG. 11A
shows a degradation plot of the all 2'-OMe oligonucleotide with
3'idT and FIG. 11B shows a degradation plot of the dRmY
oligonucleotide. The oligonucleotides were incubated at 50 nM in
95% rat plasma at 37.degree. C. and show a plasma half-life of much
greater than 48 hours for each, and that they have very similar
plasma stability profiles.
Example 7
rRmY and rGmH 2'-OMe SELEX.TM. Against Human IL-23
[0236] Selections were performed to identify aptamers containing
2'-OMe C, U and 2'-OH G, A (rRmY), and 2'-O-Methyl A, C, and U and
2'-OH G (rGmH). All selections were direct selections against human
IL-23 protein target which had been immobilized on a hydrophobic
plate. Selections yielded pools significantly enriched for h-IL-23
binding versus nave, unselected pool. Individual clone sequences
for h-IL-23 are reported herein, but h-IL-23 binding data for the
individual clones are not shown.
[0237] Pool Preparation. A DNA template with the sequence
5'-GGGAGAGGAGAGAACGTTCTACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCG
CTGTCGATCGATCGATCGATG-3' (SEQ ID NO:459) was synthesized using an
ABI EXPEDITE.TM. DNA synthesizer, and deprotected by standard
methods. The templates were amplified with the primers PB.118.95.G:
5'-GGGAGAGGAGAGAACGTTCTAC-3' (SEQ ID NO:460) and STC.104.102.A
(5'-CATCGATCGATCGATCGACAGC-3' (SEQ ID NO:461) and then used as a
template (200 nm template was used for round 1, and for subsequent
rounds approximately 50 nM, a {fraction (1/10)} dilution of an
optimized PCR reaction, using conditions described herein, was
used) for in vitro transcription with Y639F single mutant T7 RNA
polymerase. Transcriptions were done using 200 mM HEPES, 40 mM DTT,
2 mM spermidine, 0.01% TritonX-100, 10% PEG-8000, 5 mM MgCl.sub.2,
1.5 mM MnCl.sub.2, 500 .mu.M NTPs, 500 .mu.M GMP, 0.01 units/.mu.l
inorganic pyrophosphatase, and Y639F single mutant T7 polymerase.
Two different compositions were transcribed rRmY and rGmH.
[0238] Selection. Each round of selection was initiated by
immobilizing 20 pmoles of h-IL-23 to the surface Nunc Maxisorp
hydrophobic plates for 2 hours at room temperature in 100 .mu.L of
1.times.Dulbecco's PBS. The supernatant was then removed and the
wells were washed 4 times with 120 .mu.L wash buffer (1.times.DPBS,
0.2% BSA, and 0.05% Tween-20). Pool RNA was heated to 90.degree. C.
for 3 minutes and cooled to room temperature for 10 minutes to
refold. In round 1, a positive selection step was conducted.
Briefly, 1.times.10.sup.14 molecules (0.2 nmoles) of pool RNA were
incubated in 100 .mu.L binding buffer (1.times.DPBS and 0.05%
Tween-20) in the wells with immobilized protein target for 1 hour.
The supernatant was then removed and the wells were washed 4.times.
with 120 .mu.L wash buffer. In subsequent rounds a negative
selection step was included. The pool RNA was also incubated for 30
minutes at room temperature in empty wells to remove any plastic
binding sequences from the pool before the positive selection step.
The number of washes was increased after round 4 to increase
stringency. In all cases, the pool RNA bound to immobilized h-IL-23
was reverse transcribed directly in the selection plate after by
the addition of RT mix (3' primer, STC.104.102.A, and Thermoscript
RT, Invitrogen) followed by incubated at 65.degree. C. for 1 hour.
The resulting cDNA was used as a template for PCR (Taq polymerase,
New England Biolabs) "Hot start" PCR conditions coupled with a
60.degree. C. annealing temperature were used to minimize
primer-dimer formation. Amplified pool template DNA was desalted
with a Centrisep column according to the manufacturer's recommended
conditions and used to program transcription of the pool RNA for
the next round of selection. The transcribed pool was gel purified
on a 10% polyacrylamide gel every round. Table 15 shows the RNA
pool concentrations used per round of selection.
28TABLE 15 RNA pool concentrations per round of selection. pmoles
Pool rRmY PD- rGmH used 2OMe GF- 3OMe PDGF- Round IL23 hIgE mIgE BB
IL23 hIgE mIgE BB 1 200 200 200 200 200 200 200 200 2 110 140 130
135 40 50 40 60 3 65 115 60 160 100 190 90 160 4 50 40 40 30 170
120 40 240 5 80 130 130 110 100 60 40 70 6 100 80 90 39 110 140 90
90 7 50 90 130 170 70 80 130 90 8 120 190 150 60 90 110 130 9 120
210 170 80 80 100 100 10 130 210 180 11 110 210
[0239] The selection progress was monitored using a sandwich filter
binding assay. The 5'-.sup.=P-labeled pool RNA was refolded at
90.degree. C. for 3 minutes and cooled to room temperature for 10
minutes. Next, pool RNA (trace concentration) was incubated with
h-IL-23 DPBS plus 0.1 mg/ml tRNA for 30 minutes at room temperature
and then applied to a nitrocellulose and nylon filter sandwich in a
dot blot apparatus (Schleicher and Schuell). The percentage of pool
RNA bound to the nitrocellulose was calculated and monitored
approximately every 3 rounds with a signal point screen (+/-250 nM
h-IL-23). Pool K.sub.D measurements were measured using a titration
of protein and the dot blot apparatus as described above.
[0240] Selection. The rRmY h-IL-23 selection was enriched for
h-IL-23 binding vs. the nave pools after 4 rounds of selection. The
selection stringency was increased and the selection was continued
for 8 more rounds. At round 9 the pool K.sub.D was approximately
500 nM or higher. The rGmH selection was enriched over the nave
pool binding at round 10. The pool K.sub.D is also approximately
500 nM or higher. The pools were cloned using TOPO TA cloning kit
(Invitrogen) and individual sequences were generated. FIG. 12 shows
pool binding data to h-IL-23 for the rGmH round 10 and rRmY round
12 pools. Dissociation constants were estimated fitting data to the
equation: fraction RNA bound=amplitude*K.sub.D/(K.sub-
.D+[h-IL-23]). Table 16 shows the individual clone sequences for
round 12 of the rRmY selection. There is one group of 6 duplicate
sequences and 4 pairs of 2 duplicate sequences out of 48 clones.
All 48 clones will be labeled and tested for binding to 200 mM
h-IL-23. Table 17 shows the individual clone sequences for round 10
of the rGmH selection. Binding data is shown in FIG. 14.
29TABLE 16 Corresponding cDNAs of the Individual Clone Sequences
for Round 12 of the rRmY Selection. SEQ ID No.280 ARX34P2.G01
GGGAGAGGAGAGAACGTTCTACAAATGA- GAGCAGGCCGAAAAGGAGTCGC
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.281 ARX34P2.A06
GGGAGAGGAGAGAACGTTCTACAAAGGATCAATCT- TTCGGCGTATGTGTG
AGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.282 ARX34P2.E02
GGGAGAGGAGAGAACGTTCTACGGTAAAGCAGGCTGAC- TGAAAGGTTGAA
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.283 ARX34P2.H05
GGGAGAGGAGAGAACGTTCTACAGGTTAAAGCAGGCTCAGGA- ATGGAAGT
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.284 ARX34P2.G04
GGGAGAGGAGAGAACGTTCTACCAAAGCAGGCTCATAGTAATATG- GAAGT
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.285 ARX34P2.G03
GGGAGAGGAGAGAACGTTCTACAAAAGAGAGCAGGCCGAAAAGGAGTCGC
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.286 ARX34P2.N06
GGGAGAGGAGAGAACGTTCTACAAAAGGCAGGCTCAGGGGATCACTGGAA
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.287 ARX34P2.B01
GGGAGAGGAGAGAACGTTCTACAAAAAGCAGGCCGTATGGATATAAGGGA
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.288 ARX34P2.B03
GGGAGAGGAGAGAACGTTCTACAAGTGCAGGCTGCAGACATATGCGAAGT
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.289 ARX34P2.D05
GGGAGAGGAGAGAACGTTCTACAAAGGAGAGCAGGCCGAAAAGGAGTCGC
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.290 ARX34P2.C05
GGGAGAGGAGAGAACGTTCTACAAGATATAATTAAGGATAAGTGCAAAGG
AGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.291 ARX34P2.C04
GGGAGAGGAGAGAACGTTCTACAGACAACAGCNAGAGGGAATCNCANACA
AAGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.292 ARX34P2.E06
GGGAGAGAGAGAACGTTCTACAGATTCTAAGCGCAGGAATAAGTCACCAG
ACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.293 ARX34P2.A01
GGGAGAGGAGAGAACGTTCTACGAAAATGAGCATGGAAGTGGGAGTACGT
GCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.294 ARX34P2.C06
GGGAGAGGAGAGAACGTTCTACGAAGAGGCGCCGGAAGTGAGAGTAAGTG
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.295 ARX34P2.E04
GGGAGAGGAGAGAACGTTCTACGAAGTGAGTTTCCGAAGTGAGAGTACGA
ACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.296 ARX34P2.E04
GGGAGAGGAGAGAACGTTCTACGAATGAGAGCAGGCCGAAAAGGAGTCGC
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.297 ARX34P2.E04
GGGAGAGGAGAGAACGTTCTACGAGAGGCAAGAGAGAGTCGCATAAAAAA
GACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.298 ARX34P2.D06
GGGAGAGGAGAGAACGTTCTACGCAGGCTGTCGTAGACAAACGATGAAGT
CGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.299 ARX34P2.F05
GGGAGAGGAGAGAACGTTCTACGGAAAAAGATATGAAAGAAAGGATTAAG
AGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.300 ARX34P2.H02
GGGAGAGGAGAGAACGTTCTACGGAAGGNAACAANAGCACTGTTTGTGCA
GGCGCTGTCGATCNATCNATCNATGAAGGGCG SEQ ID No.301 ARX34P2.C03
GGGAGAGGAGAGAACGTTCTACGGAGCATANGGCNTGAAACTGAGANAGT
AACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.302 ARX34P2.D01
GGGAGAGGAGAGAACGTTCTACGAAAAAGGATATGAGAGAAAGGATTAAG
AGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.303 ARX34P2.A03
GGGAGAGGAGAGAACGTTCTACATACATAGGCGCCGCGAATGGGAAAGAA
AGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.304 ARX34P2.B02
GGGAGAGGAGAGAACGTTCTACTCATGAAGCCATGGTTGTAATTCTGTTT
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.305 ARX34P2.C01
GGGAGAGGAGAGAACGTTCTACTAATGCAGGCTCAGTTACTACTGGAAGT
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.306 ARX34P2.D07
GGGAGAGGAGAGAACGTTCTACTTTCATAGGCGGGATTATGGAGGAGTAT
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.307 ARX34P2.G0S
AGGAGAGGAGAGAACGTTCTACTAGAAGCAGGCTCGAATACAATTCGGAA
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.308 ARX34P2.F06
GGGAGAGGAGAGAACGTTCTACTTAGCGATGTCGGAAGAGAGAGTACGAG
GACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.309 ARX34P2.F02
GGGAGAGGAGAGAACGTTCTACTTGCGAAGACCGTGGAAGAGGAGTACTG
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.310 ARX34P2.E05
GGGAGAGGAGAGAACGTTCTACTTTTGGTGAAGGTGTAAGAGTGGCACTA
CACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.311 ARX34P2.A05
GGGAGAGGAGAGAACGTTCTACCATCAGTTGTGGCGATTATGTGGGAGTA
TGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.312 ARX34P2.E03
GGGAGAGGAGAGAACGTTCTACANAANAACATGCGATTAAAGATCATGAA
CAGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.313 ARX34P2.F04
GGGAGAGGAGAGAACGTTCTACATAAGCAGGCTCCGATAGTATTCGGGAA
GTCGCTGTCGATCGATCGATCGATGAAGGGCG
[0241]
30TABLE 17 Corresponding cDNAs of the Individual Clone Sequences
for Round 10 of the rGmH Selection. SEQ ID No.314 ARX34P2.E10
GGGAGAGGAGAGAACGTTCTACTTTCGG- AATGCGATGGGGGTGATTCGTG
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.315 ARX34P2.H09
GGGAGAGGAGAGAACGTTCTACCTGTTGAGGCTA- AGTGGATGATTGAGGG
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.316 ARX34P2.A07
GGGAGAGGAGAGAACGTTCTACCTGGGTCGGTGCGATTGGAG- ATGTCGTT
GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.317 ARX34P2.A12
GGGAGAGGAGAGAACGTTCTACCTGATGTCAGGTTGTTTGGAGAT- TATCT
GACNCTGTCNATCGATCGATCGATGAAGGGCG SEQ ID No.318 ARX34P2.A08
GGGAGAGGAGAGAACGTTCTACCTCGCGCGACGAGCGAATTTCCG- GATGC
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.319 ARX34P2.D12
GGGAGAGGAGAGAACGTTCTACCATGAATGATTGCGATCGTTGTT- CGTGT
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.320 ARX34P2.E11
GGGAGAGGAGAGAACGTTCTACTCCGACCACGCCTGGGTGATTCC- TACNA
CGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.321 ARX34P2.E12
GGGAGAGGAGAGAACGTTCTACTACTTTTGGGGATTCACTCCGCG- CTGAT
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.322 ARX34P2.D08
GGGAGAGGAGANAACGTTCTANTAGTGCTTGCGAGATAGTGTAGG- ATTAT
ACTGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.323 ARX34P2.F07
GGGAGAGGAGAGAACGTTCTACTAGTGTCCTTCTCCACGTGGTTG- TAATT
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.324 ARX34P2.B11
GGGAGAGGAGAGAACGTTCTACTATTGTGGCGCTTGTTGGACTAA- CTGAC
TACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.325 ARX34P2.F12
GGGAGAGGAGAGAACGTCCTACTTCGATTGTGATCTTGTGGCGGC- CTGTG
AGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.326 ARX34P2.A09
GGGAGAGGAGAGAACGTTCTACTTGGCGATGTCGGAAGAGAGAGT- ACGAG
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.467 ARX34P2.B07
GGGAGAGGAGAGAACGTTCTACTTGCTGTGACGGACGGGCTTGAG- AGGCT
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.327 ARX34P2.D07
GGGAGAGGAGAGAACGTTCTACTTGAANCTGCGTGAATTGANAGTAACGA
AGCGCTGTCAATCGATCNATCAATNAAGGGCG SEQ ID No.328 ARX34P2.H10
GGGAGAGGAGAGAACGTTCTACTCGAGAGGACATGTGGATCCGGTTCGCG
TGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.329 ARX34P2.H07
GGGAGAGGAGAGAACGTTCTACTGTGATGCGGTTTGCGTCGACCGGATTC
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.330 ARX34P2.F11
GGGAGAGGAGAGAACGTTCTACTGTGTGATTGGGCGCATGTCGAGGCGAC
ACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.331 ARX34P2.C07
GGGAGAGGAGAGAACGTTCTACTGATTAAGATGCGCTGGTAGAGCGGTGG
GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.332 ARX34P2.A10
GGGAGAGGAGAGAACGTTCTACTGGTTAATTTGCATGCGCGANTAACNTG
NTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.333 ARX34P2.G10
GGGAGAGGAGAGAACGTTCTACTGGGAAGCGGTAACTTGGATTCACCGAT
CCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.334 ARX34P2.H11
GGGAGAGGAGAGAACGTTCTACTGTTACGGAGATGATGGGTTTGGCTGTT
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.335 ARX34P2.C07
GGGAGAGGAGAGAACGTTCTACTTGTGGACTGAGATACGATTCGGAGCTG
GCGCTGTCGATCGATGATCGATGAAGGGCG SEQ ID No.336 AR134P2.E08
GGGAGAGGAGAGACGTTCTACTTGTGAGTTTCCTTGGGCCTTGAGCGTGG
GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.337 ARX34P2.A11
GGGAGAGGAGAGAACGTTCTACAGGTGATGTGAGCCGATTGTGAAGTTTT
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.338 ARX34P2.B08
GGGAGAGGAGAGAACGTTCTACAGCGGATGTTTGGGGGTGTGTGTTGGTT
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.339 ARX34P2.B09
GGGAGAGGAGAGAACGTTCTACATGCGGTGGTGGTCTTCGATGGGTGGAA
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.340 ARX34P2.B12
GGGAGAGGAGAGAACGTTCTACATTGGAGGGGCGCATGTGGTCTGTTTGA
TGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.341 ARX34P2.P10
GGGAGAGGAGAGAACGTTCTACGTGTTTCGCGGATTTGAAGAGGAGTAAA
ATCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.342 ARX34P2.E10
GGGAGAGGAGAGAACGTTCTACGTGTGCGTGTTCGGGAAGGGAGAGTGCC
GAGGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.343 ARX34P2.B10
GGGAGAGGAGAGAACGTTCTACGTGTGTGGTGTGCGATGCTTGGCTGTTT
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.344 ARX34P2.C08
GGGAGAGGAGAGAACGTTCTACGGTTTGTGTGGCTTGGATCTGAAGACTA
AGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.345 ARX34P2.F09
GGGAGAGGAGAGAACGTTCTACGGTTCTGGGCTTGTGTGTGAGGATTGAC
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.346 ARX34P2.C10
GGGAGAGGAGAGAACGTTCTACGATGATGAAGGCGAAAAGACGAGGCTGT
CGATCGATCGATCGATGAAGGGCG SEQ ID No.347 ARX34P2.C11
GGGAGAGGAGAGAACGTTCTACGAGTGCTGATGCGTGTCCTGGGATGGAA
TTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.348 ARX34P2.D09
GGGAGAGGAGAGAACGTTCTACGCGTTTATAGCGATCGATGATGATATAG
GCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.349 ARX34P2.D10
GGGAGAGGAGAGAACGTTCTACGCGTTCAAATGGGATAGAATTGGCTGCG
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.350 ARX34P2.D11
GGGAGAGGAGAGAACGTTCTACGAAATTGTGCGTCAGTGTGAGGCGGTTT
GCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.351 ARX34P2.E07
GGGAGAGGAGAGAACGTTCTACGGTCGAAATGAGGGTCTGGAGTTCCGAC
GACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.352 ARX34P2.E09
GGGAGAGGAGAGAACGTTCTACGAATTTGGTAATCTGGGTGACTTAGGAT
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.353 ARX34P2.G12
GGGAGAGGAGAGAACGTTCTACGATTTTTTGTGCCGAAGTAAGAGTACGC
GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.354 ARX34P2.H08
AGGAGAGGAGAGAACGTTCTACGGAGTGTGCGCGGATGAAAACAGAAGTT
GTCGCTGTCNATCGATCNATCAATGAAGGGCG
Example 8
rRmY 2'-OMe SELEX.TM. Against Human IgE
[0242] Selections were performed to identify aptamers containing
2'-OMe C, U and 2'-OH G, A (rRmY). All selections were direct
selections against human IgE protein target which had been
immobilized on a hydrophobic plate. Selections yielded pools
significantly enriched for h-IgE binding versus naive, unselected
pool. Individual clone sequences for h-IgE are reported herein, but
h-IgE binding data for the individual clones are not shown.
[0243] Pool Preparation. A DNA template with the sequence
5'-GGGAGAGGAGAGAACGTTCTACNNNNNNNNNNNNNNNNNNNNNNNNNNNNCG
CTGTCGATCGATCGATCGATG-3' (SEQ ID NO:459) was synthesized using an
ABI EXPEDITE.TM. DNA synthesizer, and deprotected by standard
methods. The templates were amplified with the primers PB.118.95.G
5'-GGGAGAGGAGAGAACGTTCTAC-3'(SEQ ID NO:460) and STC.104.102.A
5'-CATCGATCGATCGATCGACAGC-3'(SEQ ID NO:461) and then used as a
template (200 nm template was used for round 1, and for subsequent
rounds approximately 50 nM, a {fraction (1/10)} dilution of an
optimized PCR reaction, using conditions described herein, was
used) for in vitro transcription with Y639F single mutant T7 RNA
polymerase. Transcriptions were done using 200 mM HEPES, 40 mM DTT,
2 mM spermidine, 0.01% TritonX-100, 10% PEG-8000, 5 mM MgCl.sub.2,
1.5 mM MnCl.sub.2, 500 .mu.M NTPs, 500 .mu.M GMP, 0.01 units/.mu.l
inorganic pyrophosphatase, and Y639F single mutant T7 polymerase.
Selection. Each round of selection was initiated by immobilizing 20
pmoles of h-IgE to the surface Nunc Maxisorp hydrophobic plates for
2 hours at room temperature in 100 .mu.L of 1.times.Dulbecco's PBS.
The supernatant was then removed and the wells were washed 4 times
with 120 .mu.L wash buffer (1.times.DPBS, 0.2% BSA, and 0.05%
Tween-20). Pool RNA was heated to 90.degree. C. for 3 minutes and
cooled to room temperature for 10 minutes to refold. In round 1, a
positive selection step was conducted. Briefly, 1.times.10.sup.14
molecules (0.2 nmoles) of pool RNA were incubated in 100 .mu.L
binding buffer (1.times.DPBS and 0.05% Tween-20) in the wells with
immobilized protein target for 1 hour. The supernatant was then
removed and the wells were washed 4.times. with 120 .mu.L wash
buffer. In subsequent rounds a negative selection step was
included. The pool RNA was also incubated for 30 minutes at room
temperature in empty wells to remove any plastic binding sequences
from the pool before the positive selection step. The number of
washes was increased after round 4 to increase stringency. In all
cases, the pool RNA bound to immobilized h-IgE was reverse
transcribed directly in the selection plate after by the addition
of RT mix (3' primer, STC.104.102.A, and Thermoscript RT,
Invitrogen) followed by incubated at 65.degree. C. for 1 hour. The
resulting cDNA was used as a template for PCR (Taq polymerase, New
England Biolabs) "Hot start" PCR conditions coupled with a
60.degree. C. annealing temperature were used to minimize
primer-dimer formation. Amplified pool template DNA was desalted
with a Centrisep column according to the manufacturer's recommended
conditions and used to program transcription of the pool RNA for
the next round of selection. The transcribed pool was gel purified
on a 10% polyacrylamide gel every round.
[0244] rRmY pool selection against h-IgE was enriched after 4
rounds over the naive pool. The selection stringency was increased
and the selection was continued for 2 more rounds. At round 6 the
pool K.sub.D is approximately 500 nM or higher. The pools were
cloned using TOPO TA cloning kit (Invitrogen) and submitted for
sequencing. The pool contained one dominant clone
(AMX(123).A1)--which made up 71% of the clones sequenced. Three
additional clones were tested and showed a higher extent of binding
than the dominant clone. The K.sub.Ds for the pools were calculated
to be approximately 500 nM. The dissociations constants were also
calculated as described above. Table 18 shows the rRmY pool clones
after Round 6 of selection to h-IgE where the dominant clone was
AMX(123).A1 making up 40% of the 96 clones, along with 8 other
sequence families.
31TABLE 18 Corresponding cDNAs of the Individual Clone Sequence of
rRmY Pool Clones After Round 6 of Selection to h-IgE. SEQ ID No.355
ARX(123).A1
GGGAGAGGAGAGAACGTTCTACGATCTGGGCGAGCCAGTCTGACTGAGGAAGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.356 ARX34P1.B07
GGGAGAGGAGAGAACGTTCTACGAAGAAGATATGAGAGAAAGGATTAAGAGACGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.357 ARX34P1.A07
GGGAGAGGAGAGAACGTTCTACGAAAAAGATATGAGAGAAAGGATTAAGAGACGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.358 ARX34P1.A01
GGGAGAGGAGAGAACGTTCTACGAAAAAGATATGAGAGAAAGGATTAAGAGGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.359 ARX34P1.G05
GGGAGAGGAGAGAACGTTCTACGAAAAAGACATGAGAGAAAGGATTAAGAGACGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.360 ARX34P1.F09
GGGAGAGGAGAGAACGTTCTACNAAAAAGTATATGAGAGAAAGGATTAANAGACGCTGTCGATCGATCGATCG-
ATGAAGGGCG SEQ ID No.361 ARX34P1.E02
GGGAGAGGAGAGAACGTTCTACGAAAAAGATATGAGAGAAAAGGATTGAGAGATGCTGTCGATCGATCGATCG-
ATGAAGGGCG SEQ ID No.362 ARX34P1.G02
GGGAGAGGAGAGCACGTTCTACGAAAAAGATATGGAGAGAAAGGATTAAGAGACGCTGTCGATCGATCGATCG-
ATGAAGGGCG SEQ ID No.363 ARX34P1.A04
GGGAGAGGAGAGAACGTTCTACGAAAAAGATATGAGAGAAAGGATTAAAAGAGACGCTGTCATCGATCGATCG-
ATGAAGGGCG SEQ ID No.364 ARX34P1.G06
GGGAGAGGAGAGAACGTTCTACGAANAAGATACATAGTAGAAAGGATTAATAAGACGCTGTCGATCGATCGAT-
CGATGAAGGGCG SEQ ID No.365 ARX34P1.E05
GGGAGAGGAGAGAACGTTCTACAGGCGTGTTGGTAGGGTACGACGAGGCATGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.366 ARX34P1.B11
GGGAGAGGAGAGAACGTTCTACGCAAAAATGTGATGCGAGGTAATGGAACGCCGCTGTCGATCGATCGATCGA-
TTGAAGGGCG SEQ ID No.367 ARX34P1.B01
GGGAGAGGAGAGAACGTTCTACGGACCTCAGCGATAGGGGTTGAAACCGACACGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.368 ARX34P1.E06
GGGAGAGGAGAGAACGTTCTACATGGTCGGATGCTGGGGAGTAGGCAAGGTTCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.369 ARX34P1.C12
GGGAGAGGAGAGAACGTTCTACGTATCGGCGAGCGAAGCATCCGGGAGCGTTCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.370 ARX34P1.C09
GGGAGAGGAGAGAACGTTCTACGTATTGGCGCGCGAAGCATCCGGGAGCGTTCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.371 ARX34P1.A11
GGGAGAGGAGAGAACGTTCTACTTATACCTGACGGCCGGAGGCGCATAGGTGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.372 ARX34P1.H09
GGGAGAGGAGAGAACGTTCTACATGGTCGGATGCTGGGGAGTAGGCAAGGTTCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.373 ARX34P1.805
GGGAGAGGAGAGAACGTTCTACACGAGAGTACTGAGGCGCTTGGTACAGAGTCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.374 ARX34P1.B10
GGGAGAGGAGAGAACGTTCTACAGAAGGTAGAAAAAGGATAGCTGTGAGAAGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.375 ARX34P1.CO1
GGGAGAGGAGAGAACGTTCTACTGAGGGATAATACGGGTGGGATTGTCTTCCCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.376 ARX34P1.D04
GGGAGAGGAGAGAACGTTCTACATTGAGCGTTGAAGTTGGGGAAGCTCCGAGGCCGCTGTCGATCGATCGATC-
GATGAAGGGCG SEQ ID No.377 ARX34P1.E02
GGGAGAGGAGAGAACGTTCTACGCGGAGATATACAGCGAGGTAATGGAACGCCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.378 ARX34P1.F01
GGGAGAGGAGAGAACGTTCTACGAAGACAGCCCAATAGCGGCACGGAACTTGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.379 ARX34P1.G03
GGGAGAGGAGAGAACGTTCTACCGGTTGAGGGCTCGCGTGGAAGGGCCAACACGCGCTGTCGATCGATCGATC-
GATGAAGGGCG SEQ ID No.380 ARX34P1.E01
GGGAGAGGAGAGAACGTTCTACATATCAATAGACTCTTGACGTTTGGGTTTGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.381 ARX34P1.H02
GGGAGAGGAGAGAACGTTCTACAGTGAAGGAAAAGTAAGTGAAGGTGTGCGCTGTCGATCGATCGATCGATGA-
AGGGCG SEQ ID No.382 ARX34P1.H03
GGGAGAGGAGAGAACGTTCTACGGATGAAATGAGTGTCTGCGATAGGTTAAGCGCTGTCGATCGATCGATCGA-
TGAAGGGCG SEQ ID No.383 ARX34P1.N10
GGGAGAGGAGAGAACGTTCTACGGAAGGAAATGTGTGTCTGCGATAGGTTAAGCGCTGTCGATCGATCGATCG-
ATGAAGGGCG
Example 9
rRmY and rGmH 2'-OMe SELEX.TM. Against PDGF-BB
[0245] Selections were performed to identify aptamers containing
2'-OMe C, U and 2'-OH G, A (rRmY), and the other 2'-O-Methyl A, C,
and U and 2'-OH G (rGmH). All selections were direct selections
against human PDGF-BB protein target which had been immobilized on
a hydrophobic plate. Selections yielded pools significantly
enriched for h-_PDGF-BB binding versus naive, unselected pool.
Individual clone sequences for PDGF-BB are reported herein.
[0246] Pool Preparation. A DNA template with the sequence
5'-GGGAGAGGAGAGAACGTTCTACNNNNNNNNNNNNNNNNNNNNNNNNNNNNC
GCTGTCGATCGATCGATCGATG-3' (SEQ ID NO:459) was synthesized using an
ABI EXPEDITE.TM. DNA synthesizer, and deprotected by standard
methods. The templates were amplified with the primers PB.118.95.G
5'-GGGAGAGGAGAGAACGTTCTAC-3' (SEQ ID NO:460) and STC.104.102.A
5'-CATCGATCGATCGATCGACAGC-3'(SEQ ID NO:461) and then used as a
template (200 nm template was used for round 1, and for subsequent
rounds approximately 50 nM, a {fraction (1/10)} dilution of an
optimized PCR reaction, using conditions described herein, was
used) for in vitro transcription with Y639F single mutant T7 RNA
polymerase. Transcriptions were done using 200 mM HEPES, 40 mM DTT,
2 mM spermidine, 0.01% TritonX-100, 10% PEG-8000, 5 mM MgCl.sub.2,
1.5 mM MnCl.sub.2, 500 .mu.M NTPs, 500 .mu.M GMP, 0.01 units/.mu.l
inorganic pyrophosphatase, and Y639F single mutant T7 polymerase.
Two different compositions were transcribed rRmY and rGmH.
Selection. Each round of selection was initiated by immobilizing 20
pmoles of PDGF-BB to the surface Nunc Maxisorp hydrophobic plates
for 2 hours at room temperature in 100 .mu.L of 1.times.Dulbecco's
PBS. The supernatant was then removed and the wells were washed 4
times with 120 .mu.L wash buffer (1.times.DPBS, 0.2% BSA, and 0.05%
Tween-20). Pool RNA was heated to 90.degree. C. for 3 minutes and
cooled to room temperature for 10 minutes to refold. In round 1, a
positive selection step was conducted. Briefly, 1.times.10.sup.14
molecules (0.2 nmoles) of pool RNA were incubated in 100 .mu.L
binding buffer (1.times.DPBS and 0.05% Tween-20) in the wells with
immobilized protein target for 1 hour. The supernatant was then
removed and the wells were washed 4.times. with 120 .mu.L wash
buffer. In subsequent rounds a negative selection step was
included. The pool RNA was also incubated for 30 minutes at room
temperature in empty wells to remove any plastic binding sequences
from the pool before the positive selection step. The number of
washes was increased after round 4 to increase stringency. In all
cases, the pool RNA bound to immobilized PDGF-BB was reverse
transcribed directly in the selection plate after by the addition
of RT mix (3' primer, STC.104.102.A, and Thermoscript RT,
Invitrogen) followed by incubated at 65.degree. C. for 1 hour. The
resulting cDNA was used as a template for PCR (Taq polymerase, New
England Biolabs) "Hot start" PCR conditions coupled with a
60.degree. C. annealing temperature were used to minimize
primer-dimer formation. Amplified pool template DNA was desalted
with a Centrisep column according to the manufacturer's recommended
conditions and used to program transcription of the pool RNA for
the next round of selection. The transcribed pool was gel purified
on a 10% polyacrylamide gel every round.
[0247] Although the nave pool does bind to PDGF-BB, the rRmY
PDGF-BB selection was enriched after 4 rounds over the naive pool.
The selection stringency was increased and the selection was
continued for 8 more rounds. At round 12 the pool is enriched over
the nave pool, but the K.sub.D is very high. The rGmH selection was
enriched over the naive pool binding at round 10. The pool K.sub.D
is also approximately 950 nM or higher. The pools were cloned using
TOPO TA cloning kit (Invitrogen) and submitted for sequencing.
After 12 rounds of PDGF-BB pool selection clones were transcribed
and sequenced. Table 19 shows the clone sequences. FIG. 13(A) shows
a binding plot of round 12 pools for rRmY pool PDGF-BB selection
and FIG. 13(B) shows a binding plot of round 10 pools for rGmH pool
PDGF-BB selection. Dissociation constants were again measured using
the sandwich filter binding technique. Dissociation constants
(K.sub.Ds) were estimated fitting the data to the equation:
fraction RNA bound=amplitude*K.sub.D/(K.sub.D+[PDGF-BB]).
32TABLE 19 Corresponding cDNAs of the Individual Clone Sequence of
rRmY Pool Clones After Round 12 of Selection to PDGF-BB. SEQ ID
No.384 PDGF-BB ARX36.SCK.E05
GGGAGAGGAGAGAACGTTCTACATCCTTGCGTATGATCGGCATCGTAAGA
CACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.385 PDGF-BB
ARX36.SCK.F05 GGGAGAGGAGAGAACGTTCTACATCCTTGCGTATGATCGGCA- TCGTAAGA
CACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.386 PDGF-BB
ARX36.SCK.E01 GGGAGAGGAGAGAACGTTCTACGATCGAAGTCG- TGACAGAAACCACTCGC
TGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.387 PDGF-BB ARX36.SCK.F01
GGGAGAGGAGAGAACGTTCTACGATCGAAG- TCGTGACAGAAACCACTCGC
TGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.388 PDGF-BB ARX36.SCK.G01
GGGAGAGGAGAGAACGTTCTACGGAA- AAGGTTGGCGAAACGAAGAAGAAT
TTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.389 PDGF-BB
ARX36.SCK.G02 GGGAGAGGAGAGAACGTTCTACGGAAAAGGTTGGCGAAACGAAGAANAAT
TTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.390 PDGF-BB
ARX36.SCK.F04 GGGAGAGGAGAGAACGTTCTACTGGGAGTTGCGGTGTTTTGCGGTGGATT
TGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.391 PDGF-BB
ARX36.SCK.E04 GGGAGAGGAGAGAACGTTCTACTGGGAGTTGCGGTGTTTTGC- GGTGGATT
TGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.392 PDGF-BB
ARX36.SCK.F02 GGGAGAGGAGAGAACGCTCTACAAGATTGTAGA- TCAACAGCGAAGGCGTG
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.393 PDGF-BB
ARX36.SCK.E02 GGGAGAGGAGAGAACGCTCTACAAGA- TTGTAGATCAACAGCGAAGGCGTG
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.394 PDGF-BB
ARX36.SCK.A02 GGGAGAGGAGAGAACGTTCTACAAANAAGATNNCCANCNNGAGANAAAGG
AGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.395 PDGF-BB
ARX36.SCK.A03 GGGAGAGGAGAGAACGTTCTACAAACATCGAAGATCGAACTGAAAAGGAG
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.396 PDGF-BB
ARX36.SCK.A06 GGGAGAGGAGAGAACGTTCTACATGTGCATGCAAGGTGGGGC- TGACACGA
GCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.397 PDGF-BB
ARX36.SCK.B01 GGGAGAGGAGAGAACGTTCTACAAGGAGTAGAT- CGACAGAATAGAAAAAT
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.398 PDGF-BB ARX36.SCK.B02
GGGAGAGGAGAGAACGTTCTACAAAA- GGTAAGGTCAAAAAAGCGCAACGT
TGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.399 PDGF-BB
ARX36.SCK.D04 GGGAGAGGAGAGAACGTTCTACAAAAGGAGGCGAAATAAGTGAGACAATG
TGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.400 PDGF-BB
ARX36.SCK.B04 GGGAGAGGAGAGAACGTTCTACAAAAATCCACAAACATAGCTGTAATTGC
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.401 PDGF-BB ARX36.SCK.B05
GGGAGAGGAGAGACGTTCTACAAGAACATATAACATTTTGGT- TGAGAGCA
ACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.402 PDGF-BB ARX36.SCK.D03
GGGAGAGAGAGAACGTTCTACAAGAGTCNACGA- TTTCNATCACAAATGTG
GCTGCTGTCNATCGATCGATCNATGAAGGGCG SEQ ID No.403 PDGF-BB
ARX36.SCK.C01 GGGAGAGGAGAGAACGTTCTACAAGC- AAGCAAAAAAAGTATCGACAGAAG
TGGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.404 PDGF-BB
ARX36.SCK.D06 GGGAGAGGAGAGAACGTTCTACAAGTAATATCAGAGCAATCGGAATAAGA
GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.405 PDGF-BB
ARX36.SCK.D02 GGGAGAGGAGAGAACGTTCTACAGACTTCGATGCGATGGATTTGGAAATG
TGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.406 PDGF-BB
ARX36.SCK.C03 GGGAGAGGAGAGAACGTTCTACAGAAAGAATTACAGGAACAA- ATACACGT
GCGGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.407 PDGF-BB
ARX36.SCK.F06 GGGAGAGGAGAGAACGTTCTACAGAAATCAATC- GAGGTGATCGTTATATA
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.408 PDGF-BB
ARX36.SCK.C04 GGGAGAGGAGAGAACGTTCTACAGAT- TTGGATCGACAATCTCGTAGAAGA
GACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.409 PDGF-BB
ARX36.SCK.C06 GGGAGAGGAGAGAACGTTCTACAATGCAAGTTTAAGTGTGGTGTCAAACG
CACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.410 PDGF-BB
ARX36.SCK.G03 GGGAGAGGAGAGAACGTTCTACAAATAAAGACACGAAGATCGACGGAGAC
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.411 PDGF-BB ARX36.SCK.F03
GGGAGAGGAGAGAACGTTCTACGAAGATGTGTTTAAGAATCG- AGGTTTTC
GACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.412 PDGF-BB
ARX36.SCK.C02 GGGAGAGGAGAGAACGTTCTACGAGTTGGCACG- CATGTATAGGTATTTTG
GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.413 PDGF-BB ARX36.SCK.B03
GGGAGAGGAGAGAACGTTCTACGAAA- AAAAGAGATGAGAGAAAGGATTAA
GAGACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.414 PDGF-BB
ARX36.SCK.B06 GGGAGAGGAGAGAACGTTCTACGAAAAGGAAAAAAAACGATCGGCAGAGT
CCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.415 PDGF-BB
ARX36.SCK.C05 GGGAGAGGAGAGAACGTTCTACGATTAAGGAAACATTTACGCGAATACAT
GACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.416 PDGF-BB
ARX36.SCK.D01 GGGAGAGGAGAGAACGTTCTACGACGTTTGCTCTGAAAATAG- GACAGAAG
GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.417 PDGF-BB ARX36.SCK.E03
GGGAGAGGAGAGAACGTTCTACGAAGATGTGTT- TAAGAATCGAGGTTTTC
GACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.418 PDGF-BB
ARX36.SCK.A04 GGGAGAGGAGAGAACGTTCTACCGAG- ATCGAAAGGTAAGAGAAAATTCAT
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.419 PDGF-BB
ARX36.SCK.A05 GGGAGAGGAGAGAACGTTCTACTAAGATTCGTCGTTCAGACAGAGAAAGC
GACGCTGTCGATCGATCGATCGATGAAGGGCG
[0248]
33TABLE 20 Corresponding cDNAs of the Individual Clone Sequence of
rGmH Pool Clones After Round 10 of Selection to PDGF-BB. SEQ ID
No.420 PDGF-BB ARX36.SCK.E08.M13F
GGGAGAGGAGAGAACGTTCTACCTTGGCGACGATCTGTGACCTGAA- TTTT
TGTCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.421 PDGF-BB
ARX36.SCK.F08.M13F GGGAGAGGAGAGAACGTTCTACCTTGGC-
GACGATCTGTGACCTGAATTTT TGTCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.422 PDGF-BB ARX36.SCK.E09.M13F
GGGAGAGGAGAGAACGTTCTACCTTGGTCTCAGCAGCTTTTAACAAAGTA
TCCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.423 PDGF-BB
ARX36.SCK.F09.M13F GGGAGAGGAGAGAACGTTCTACCTTGGTCTCAGCAGCTTTTAACA-
AAGTA TCCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.424 PDGF-BB
ARX36.SCK.F07.M13F GGGAGAGGAGAGAACGTTCTACCGCTAT-
TTTGTTCATTGAAGGACTTGTC ACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.425 PDGF-BB ARX36.SCK.E07.M13F
GGGAGAGGAGAGAACGTTCTACCGCTATTTTGTTCATTGAAGGACTTGTC
ACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.426 PDGF-BB
ARX36.SCK.E11.M13F GGGAGAGGAGAGAACGTTCTACCCTATTGAGGTTGATTGGAAGTGCC-
TAT GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.427 PDGF-BB
ARX36.SCK.F11.M13F GGGAGAGGAGAGAACGTTCTACCCTATTGAGGTTGA-
TTGGAAGTGCCTAT GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.428
PDGF-BB ARX36.SCK.F10.M13F GGGAGAGGAGAGAACGTTCTACTGA-
AGATGTTATGATGATTGACGAGGAG GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.429 PDGF-BB ARX36.SCK.E10.M13F
GGGAGAGGAGAGAACGTTCTACTGAAGATGTTATGATGATTGACGAGGAG
GCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.430 PDGF-BB
ARX36.SCK.E12.M13F GGGAGAGGAGAGAACGTTCTACTGTCTGAGTGTCGCCGCCTTGTGTG-
ATG TTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.431 PDGF-BB
ARX36.SCK.F12.M13F GGGAGAGGAGAGAACGTTCTACTGTCTGAGTGTCGC-
CGCCTTGTGTGATG TTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.432
PDGF-BB ARX36.SCK.A07.M13F GGGAGAGGAGAGAACGTTCTACGTG-
ATGGCTGTGAATGAGGTAGTTCGAA TACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.433 PDGF-BB ARX36.SCK.C12.M13F
GGGAGAGGAGAGAACGTTCTACGTGAAATCAAGGTTGTTAATTTGGGGAA
TCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.434 PDGF-BB
ARX36.SCK.B07.M13F GGGAGAGGAGAGAACGTTCTACGTATAAGGCCGTAACCGGGTAGCGA-
GTG GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.435 PDGF-BB
ARX36.SCK.A09.M13F GGGAGAGGAGAGAACGTTNTACGTGGGCGAAGGAGC-
TGCGGGCGTTGNAG TTTGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.436
PDGF-BB ARX36.SCK.A11.M13F GGGAGAGGAGAGAACGTTCTACGTC-
ATCCTAGTCTGAGATCGGATTTTCT TGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.437 PDGF-BB ARX36.SCK.C09.M13F
GGGAGAGGAGAGAACGTTCTACGTTTGCGAGTGTGGTCGACGCTGAATGC
GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.438 PDGF-BB
ARX36.SCK.A08.M13F GGGAGAGGAGAGAACGTTCTACGGATTGATAGGGATTGAGATGAGG-
TCTT GTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.439 PDGF-BB
ARX36.SCK.D07.M13F GGGAGAGGAGAGAACGTTCTACGATGTCGTGTTAG-
ATTACTTATTGCTAT CTGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.440
PDGF-BB ARX36.SCK.D08.M13F GGGAGAGGAGAGAACGTTCTACGAT-
GCCTGGCGGAAACGGAGCCTGGGAT TTCGCTGTCNATCGATCGATCGATGAAGGGCG SEQ ID
No.441 PDGF-BB ARX36.SCK.B11.M13F
GGGAGAGGAGAGAACGTTCTACGAGGATTTGACGTGTGTGTGCTAGAGTA
CGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.442 PDGF-BB
ARX36.SCK.D09.M13F GGGAGAGGAGAGAACGTTCTACGAGTATTATGCGTCCCTTGAGGATA-
CAC GGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.443 PDGF-BB
ARX36.SCK.B10.M13F GGGAGAGGAGAGAACGTTCTACAGGGATAACTGTAG-
CGATGAAAGTAAAC GATGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.444
PDGF-BB ARX36.SCK.C10.M13F GGGAGAGGAGAGAACGTTCTACAAG-
AAGTGTGGCCGCAGAGACGAAATGC ACGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.445 PDGF-BB ARX36.SCK.A10.M13F
GGGAGAGGAGAGAACGTTCTACCCATATCTTCCTTCTTTATTCCGTTAGT
TGCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.446 PDGF-BB
ARX36.SCK.B09.M13F GGGAGAGGAGAGAACGTTCTACCTGTGTTGATGCTTCCGTTTGAG-
ATTGC CCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.447 PDGF-BB
ARX36.SCK.B12.M13F GGGAGAGGAGAGAACGTTCTACCNGTAA-
GANAANCTATTTTAGCCCTTGN NCTGCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.448 PDGF-BB ARX36.SCK.C08.M13F
GGGAGAGGAGAGAACGTTCTACCCTTGTCCTCCAATCCTCTTTTGACTCT
TGCCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.449 PDGF-BB
ARX36.SCK.D12.M13F GGGAGAGGAGAGAACGTTCTACCTGATTTTGTCACTGGATTCCGA-
TGGCT TTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.450 PDGF-BB
ARX36.SCK.C11.M13F GGGAGAGGAGAGAACGTTCTACTGTAAT-
AAGGGATGCGTCAGGAACCTGT GTTCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID
No.451 PDGF-BB ARX36.SCK.D11.M13F
GGGAGAGGAGAGAACGTTCTACTGCTTTCCGGGAATTTGTTTGTTTGCTT
CCGCTGTCGATCGATCGATCGATGAAGGGCG SEQ ID No.452 PDGF-BB
ARX36.SCK.C07.M13F GGGAGAGGAGAGAACGTTCTACTTCGTCGGTTCACTTTTCTTCGTGT-
AGT GTCGCTGTCGATCGATTGATCGATGAAGGGCG SEQ ID No.189 PDGF-BB
ARX36.SCK.A12.M13F GGGAGAGGAGAGAACGTTCTACTATGAAGGGTTTTA-
AAGATGACACATTA GCCGCTGTCGATCGATCGATCGATGAAGGGCG
[0249] The present invention having been described by detailed
description and the foregoing non-limiting examples, is now defined
by the spirit and scope of the following claims.
Sequence CWU 1
1
468 1 93 DNA Artificial aptamer library template ARC254 1
catcgatgct agtcgtaacg atccnnnnnn nnnnnnnnnn nnnnnnnnnn nnnncgagaa
60 cgttctctcc tctccctata gtgagtcgta tta 93 2 92 DNA Artificial
aptamer library template ARC255 2 catgcatcgc gactgactag ccgnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnngtagaac 60 gttctctcct ctccctatag
tgagtcgtat ta 92 3 77 DNA Artificial clone of aptamer 3 gggagaggag
agaacgttct cgaaatgatg catgttcgta aaatggcagt attggatcgt 60
tacaactagc atcgatg 77 4 76 DNA Artificial clone of aptamer 4
gggagaggag agaacgttct cgtgccgagg tccggaacct tgatgattgg cgggatcgtt
60 acgactagca tcgatg 76 5 76 DNA Artificial clone of aptamer 5
gggagaggag agaacgttct cgcatttggg ctagttgtga aatggcagta ttggatcgtt
60 acgactagca tcgatg 76 6 76 DNA Artificial clone of aptamer 6
gggagaggag agaacgttct cgaatcgtag atagtcgtga aatggcagta ttggatcgtt
60 acgactagca tcgatg 76 7 76 DNA Artificial clone of aptamer 7
gggagaggag agaacgttct cgttctagtc ggtacgatat gttgacgaat ccggatcgtt
60 acgactagca tcgatg 76 8 78 DNA Artificial clone of aptamer 8
gggagaggag agaacgttct cgtttgatga ggcggacata atccgtgccg agcgggatcg
60 ttacgactag catcgatg 78 9 77 DNA Artificial clone of aptamer 9
gggagaggag agaacgttct cgaaggaaaa gagtttagta ttggccgtcc gtgggatcgt
60 tacgactagc atcgatg 77 10 76 DNA Artificial clone of aptamer 10
gggagaggag agaacgttct cgtgccgagg tccggaacct tgatgattgg cgggatcgtt
60 acgactagca tcgatg 76 11 76 DNA Artificial clone of aptamer 11
gggagaggag agaacgttct cgtacggtcc attgagtttg agatgtcgcc atggatcgtt
60 acgactagca tcgatg 76 12 77 DNA Artificial clone of aptamer 12
gggagaggag agaacgttct cgagttagtg gtaactgata tgttgaattg tccggatcgt
60 tacgactagc atcgatg 77 13 76 DNA Artificial clone of aptamer 13
gggagaggag agaacgttct cgcacggatg gcgagaacag agattgctag gtggatcgtt
60 acgactagca tcgatg 76 14 76 DNA Artificial clone of aptamer 14
gggagaggag agaacgttct cgntancgnt ncgccntgct aacgcntant tgggatcgtt
60 acgactagca tcgatg 76 15 77 DNA Artificial clone of aptamer 15
gggagaggag agaacgttct cgaagatgag ttttgtcgtg aaatggcagt attggatcgt
60 tacgactagc atcgatg 77 16 76 DNA Artificial clone of aptamer 16
gggagaggag agaacgttct cgggatgccg gattgatttc tgatgggtac tgggatcgtt
60 acgactagca tcgatg 76 17 76 DNA Artificial clone of aptamer 17
gggagaggag agaacgttct cgaatggaat gcatgtccat cgctagcatt tgggatcgtt
60 acgactagca tcgatg 76 18 76 DNA Artificial clone of aptamer 18
gggagaggag agaacgttct cgtgctgagg tccggaacct tgatgattgg cgggatcgtt
60 ncnactagca tcgatg 76 19 76 DNA Artificial clone of aptamer 19
gggagaggag agaacgttct cgctaattgc tgagtcgtga agtggcagta ttggatcgtt
60 acgactagca tcgatg 76 20 76 DNA Artificial clone of aptamer 20
gggagaggag agaacgttct cgtaacgatg tccggggcga aaggctagca tgggatcgtt
60 acgactagca tcgatg 76 21 77 DNA Artificial clone of aptamer 21
gggagaggag agaacgttct cgatgcgatt gtcgagattt gtaagatagc tgtggatcgt
60 tacgactagc atcgatg 77 22 76 DNA Artificial clone of aptamer 22
gggagaggag agaacgttct cgcagaaaac atctttgcgg ttgaatacat gtggatcgtt
60 acgactagca tcgatg 76 23 76 DNA Artificial clone of aptamer 23
gggagaggag agaacgttct cgaaaaaaga nancnncctt cngaatacat gcggatcgtt
60 acgactagca tcgatg 76 24 76 DNA Artificial clone of aptamer 24
gggagaggag agaacgttct cgagagtgat tcgatgcttc angaatacat gtggatcgtt
60 acgactagca tcgatg 76 25 81 DNA Artificial clone of aptamer 25
gggagaggag agaacgttct cgacannncn tngctnggtt gantacatgt gnntntcnnn
60 ancnntnntc tntnanaggg g 81 26 76 DNA Artificial clone of aptamer
26 gggagaggag agaacgttct cgaagaagga aagctgcaag tcgaatacac
gcggatcgtt 60 acgactagca tcgatg 76 27 76 DNA Artificial clone of
aptamer 27 gggagaggag agaacgttct cgcaaaaaca tcgattacag ttgagtacat
gtggatcgtt 60 acgactagca tcgatg 76 28 73 DNA Artificial clone of
aptamer 28 gggagaggag agaacgttct cgagacatca ttgctcgttg aatacatgtg
gatcgttacg 60 actagcatcg atg 73 29 76 DNA Artificial clone of
aptamer 29 gggagaggag agaacgttct cgccaaagta gcttcgacag tcgaatacat
gtggatcgtt 60 acgactagca tcgatg 76 30 76 DNA Artificial clone of
aptamer 30 gggagaggag agaacgttct cgaaaatcag tactgtgcag tcgaatacat
gcggatcgtt 60 acgactagca tcgatg 76 31 76 DNA Artificial clone of
aptamer 31 gggagaggag agaacgttct cgtaatgaca tcaatgcttc ttgaatacag
gtggatcgtt 60 acgactagca tcgatg 76 32 75 DNA Artificial clone of
aptamer 32 gggagaggag agaacgttct cgagaaaaac gatctgtgac gtgtaatccg
cggatcgtta 60 cgactagcat cgatg 75 33 76 DNA Artificial clone of
aptamer 33 gggagaggag agaacgttct cgcaacaaac gtcgacgctt ctgaatacat
gtggatcgtt 60 acgactagca tcgatg 76 34 76 DNA Artificial clone of
aptamer 34 gggagaggag agaacgttct cgtgatcata gaaatgctag ctgaatacat
gtggatcgtt 60 acgactagca tcgatg 76 35 75 DNA Artificial clone of
aptamer 35 gggagaggag agaacgttct cgcagcgtaa aatgcttttc gaagtacatg
tggatcgtta 60 cgactagcat cgatg 75 36 76 DNA Artificial clone of
aptamer 36 gggagaggag agaacgttct cgccaagaat caatcgcttg tcgaatacat
gcggatcgtt 60 acgactagca tcgatg 76 37 76 DNA Artificial clone of
aptamer 37 gggagaggag agaacgttct cgtgatcata gaaatgctag ctgagtacat
gtggatcgtt 60 acgactagca tcgatg 76 38 76 DNA Artificial clone of
aptamer 38 gggagaggag agaacgttct cgcagaaaac atctttgcgg ttgaatacat
gtggatcgtt 60 acgactagca tcgatg 76 39 78 DNA Artificial clone of
aptamer 39 gggagaggag agaacgttct cgnaaacann catctattgn agttgaatac
atgtggatcg 60 ttacgactag catcgatg 78 40 76 DNA Artificial clone of
aptamer 40 gggagaggag agaacgttct cgctaaagat tcgctgcttg ccgaatacat
gtggatcgtt 60 acgactagca tcgatg 76 41 76 DNA Artificial clone of
aptamer 41 gggagaggag agaacgttct cgggttttgt ctgcgtttgt gcgttgaacc
cgggatcgtt 60 acgactagca tcgatg 76 42 77 DNA Artificial clone of
aptamer 42 gggagaggag agaacgttct cgtgattacg tgatgaggat ccgcgttttc
tcgggatcgt 60 tacgactagc atcgatg 77 43 76 DNA Artificial clone of
aptamer 43 gggagaggag agaacgttct cgttagtgaa aacgatcatg catgtggatc
gcggatcgtt 60 acgactagca tcgatg 76 44 75 DNA Artificial clone of
aptamer 44 gggagaggag agaacgttct cgtgttcatt cgtttgctta tcgttgcatg
tggatcgtta 60 cgactagcat cgatg 75 45 76 DNA Artificial clone of
aptamer 45 aggagaggag agaacgttct cggcagagtg tgatgtgcat ccgcacgtgc
cgggatcgtt 60 acgactagca tcgatg 76 46 76 DNA Artificial clone of
aptamer 46 gggagaggag agaacgttct cgttagtaaa tacgatcgtg catgtggatc
gcggatcgtt 60 acgactagca tcgatg 76 47 77 DNA Artificial clone of
aptamer 47 gggagaggag agaacgcccc cctgattncg tgaagaggat ccgcantttc
ncgggatcgt 60 tacgactagc atcgatg 77 48 76 DNA Artificial clone of
aptamer 48 gggagaggag agaacgttct cgtggctttg gaacgggtac ggatttggca
cgggatcgtt 60 acgactagca tcgatg 76 49 77 DNA Artificial clone of
aptamer 49 gggagaggag agaacgttct cgtgattacg tgatgaggat ccgcgttttc
tcgggatcgt 60 tacgactagc atcgatg 77 50 76 DNA Artificial clone of
aptamer 50 gggagaggag agaacgttct cgtcattggt gacngcgttg catgtggatc
gcggatcgtt 60 acgactagca tcgatg 76 51 76 DNA Artificial clone of
aptamer 51 gggagaggag agaacgttct cgntggtnna angcttttgt ngggntannt
gtggatcgtt 60 acgactagca tcgatg 76 52 76 DNA Artificial clone of
aptamer 52 gggagaggag agaacgttct cgtggctttg gaacgaattc ggatttggca
cgggatcgtt 60 acgactagca tcgatg 76 53 75 DNA Artificial clone of
aptamer 53 gggagaggag agaacgttct cgtgcgatgt cgtggatttc cgtttcgcaa
gggatcgtta 60 cgactagcat cgatg 75 54 76 DNA Artificial clone of
aptamer 54 gggagaggag agaacgttct cgtgaagcag atgtcgttgg cgacttagag
ggggatcgtt 60 acgactagca tcgatg 76 55 77 DNA Artificial clone of
aptamer 55 gggagaggag agaacgttct cgtgatttcg tgatgaggat ccgcgttttc
tcgggatcgt 60 tacgactagc atcgatg 77 56 75 DNA Artificial clone of
aptamer 56 gggagaggag agaacgttct cgctagtaac gatgacttga tgagcatccg
aggatcgtta 60 cgactagcat cgatg 75 57 76 DNA Artificial clone of
aptamer 57 gggagaggag agaacgttct cgtcataagt aacgacgttg catgtggatc
gcggatcgtt 60 acgactagca tcgatg 76 58 76 DNA Artificial clone of
aptamer 58 gggagaggag agaacgttct cgcaaggaga tggttgctag ctgagtacat
gtggatcgtt 60 acgactagca tcgatg 76 59 78 DNA Artificial clone of
aptamer 59 gggagaggag agaacgttct cgcgatatgc agtttgagaa gtcgcgcatt
cgggggatcg 60 ttacgactag catcgatg 78 60 75 DNA Artificial clone of
aptamer 60 gggagaggag agaacgttct cgtgcgacgg gcttcttgtg tcattcgcat
gggatcgtta 60 cgactagcat cgatg 75 61 76 DNA Artificial clone of
aptamer 61 gggagaggag agaacgttct cggcattgca gttgataggt cgcgcagtgc
tgggatcgtt 60 acgactagca tcgatg 76 62 78 DNA Artificial clone of
aptamer 62 gggagaggag agaacgttct cgcgatatgc agtctgagaa gtcgcgcatt
cgagggatcg 60 ttacgactag catcgatg 78 63 76 DNA Artificial clone of
aptamer 63 gggagaggag agaacgttct cgtgtagcaa gcatgtggat cgcgactgca
cgggatcgtt 60 acgactagca tcgatg 76 64 76 DNA Artificial clone of
aptamer 64 gggagaggag agaacgttct cggataagca gttgagatgt cgcgctttga
cgggatcgtt 60 acgactagca tcgatg 76 65 75 DNA Artificial clone of
aptamer 65 gggagaggag agaacgttct cgatgancan tttgagaagt cgcgcttgtc
gggatcgtta 60 cgactagcat cgatg 75 66 75 DNA Artificial clone of
aptamer 66 gggagaggag agaacgttct cgagtaatgc agtggaagtc gcgcattacc
tgggatcgtt 60 acgactagca tcatg 75 67 78 DNA Artificial clone of
aptamer 67 gggagaggag agaacgttct cgcgatatgc agtttgagaa gtcgcgcatt
cgggggatcg 60 ttacgactag catcgatg 78 68 73 DNA Artificial clone of
aptamer 68 gggagaggag agaacgttct cgtgatncag ttganaagtc ncgcatacag
gatcgttacg 60 actagcatcg atg 73 69 76 DNA Artificial clone of
aptamer 69 gggagaggag agaacgttct cgagtaatgc tgtggaagtc gcgcatttcc
tgggatcgtt 60 acgactagca tcgatg 76 70 76 DNA Artificial clone of
aptamer 70 gggagaggag agaacgttct cggcattgca gttgataggt cgcgcagtgc
tgggatcgtt 60 acgactagca tcgatg 76 71 78 DNA Artificial clone of
aptamer 71 gggagaggag agaacgttct cgcgatatgc agtttgggaa gtcgcgcatt
cgagggatcg 60 ttacgactag catcgatg 78 72 78 DNA Artificial clone of
aptamer 72 gggagaggag agaacgttct cgcnatatgc tgtttganaa ntcgcgcatt
cgggggatcg 60 ttacgactag catcgatg 78 73 78 DNA Artificial clone of
aptamer 73 gggagaggag agaacgttct cgcgtagatt gggctgaatg ggatatcttt
agcgggatcg 60 ttacgactag catcgatg 78 74 78 DNA Artificial clone of
aptamer 74 gggagaggag agaacgttct cgcgatatgc agtttgagaa gtcgcgcttt
cgagggatcg 60 ttacgactag catcgatg 78 75 78 DNA Artificial clone of
aptamer 75 gggagaggag agaacgttct cgtcaatctg atgtagcctc acgtgggcgg
agtcggatcg 60 ttacgactag catcgatg 78 76 45 DNA Artificial clone of
aptamer 76 gggagaggag agaacgttct cggatcgtta cgactagcat cgatg 45 77
45 DNA Artificial clone of aptamer 77 gggagaggag agaacgttct
cggatcgtta cgactagcat cgatg 45 78 76 DNA Artificial clone of
aptamer 78 gggagaggag agaacgttct cggtggtgtt gctgaactgt cgcgtttcgc
cgggatcgtt 60 acgactagca tcgatg 76 79 77 DNA Artificial clone of
aptamer 79 gggagaggag agaacgttct cgtcgcgatt gcatattttc cgccttgctg
tgaggatcgt 60 tacgactagc atcgatg 77 80 78 DNA Artificial clone of
aptamer 80 gggagaggag agaacgttct cgcgatttgc agtttgagat gtcgcgcatt
cgagggatcg 60 ttacgactag catcgatg 78 81 78 DNA Artificial clone of
aptamer 81 gggagaggag agaacgttct cgcgatatgc agtttgagaa gtcgcgcatt
cgggggatcg 60 ttacgactag catcgatg 78 82 76 DNA Artificial clone of
aptamer 82 gggagaggag agaacgttct cgttggtgca gtttgagatg tcgcgcacct
tgggatcgtt 60 acgactagca tcgatg 76 83 80 DNA Artificial clone of
aptamer 83 gggagaggag agaacgttct cggtattggt tccattaagc tggacactct
gctccgggat 60 cgttacgact agcatcgatg 80 84 76 DNA Artificial clone
of aptamer 84 gggagaggag agaacgttct cgttggtgca gtttgagatg
tcgcgcgcct tgggatcgtt 60 acgactagca tcgatg 76 85 78 DNA Artificial
clone of aptamer 85 gggagaggag agaacgttct cgcgatatgc agtttgagaa
gtcgcgcatt cgagggatcg 60 ttacnactag catcgatg 78 86 78 DNA
Artificial clone of aptamer 86 gggagaggag agaacgttct cgcgatatgc
agtttgagaa gtcgcgcatt cgggggatcg 60 ttacgactag catcgatg 78 87 80
DNA Artificial clone of aptamer 87 gggagaggag agaacgctct cggggacnna
aanncgaatt gncgcgtgng tccgggggag 60 cgcccgacta gtcatcgatg 80 88 78
DNA Artificial clone of aptamer 88 gggagaggag agaacgttct cgcgatatgn
antttgagaa gtcgcgcatt cgggggatcg 60 ttacgactag catcgatg 78 89 75
DNA Artificial clone of aptamer 89 gggagaggag agaacgttct cggtgtacag
cttgagatgt cgcgtactcc gggatcgtta 60 cgactagcat cgatg 75 90 78 DNA
Artificial clone of aptamer 90 gggagaggag agaacgttct cgcgatatgc
agtttgagaa gtcgcgcatt cgggggatcg 60 ttacgactag catcgatg 78 91 76
DNA Artificial clone of aptamer 91 gggagaggag agaacgttct cgagtaagaa
agctgaatgg tcgcacttct cgggatcgtt 60 acgactagca tcgatg 76 92 78 DNA
Artificial clone of aptamer 92 agggagagga agaacgttct cgcgatgtgc
agtttgagaa gtcgcgcatt cgagggatcg 60 ttacgactag catcgatg 78 93 76
DNA Artificial clone of aptamer 93 gggagaggag agaacgttct cgaaagaatc
agcatgcgga tcgcggcttt cgggatcgtt 60 acgactagca tcgatg 76 94 79 DNA
Artificial clone of aptamer 94 gggagaggag agaacgttct cgantccant
ntncntggag gagtaagtac ctgagggatc 60 gttacgacta gcatcgatg 79 95 76
DNA Artificial clone of aptamer 95 gggagaggag agaacgttct cgggaaacaa
ggaacttaga gttanttgac cgggatcgtt 60 acgactagca tcgatg
76 96 76 DNA Artificial clone of aptamer 96 gggagaggag agaacgttct
cgtaccatgc aaggaacata atagttagcg tgggatcgtt 60 acgactagca tcgatg 76
97 76 DNA Artificial clone of aptamer 97 gggagaggag agaacgttct
cgggacacaa ggaacacaat agttagtgta cgggatcgtt 60 acgactagca tcgatg 76
98 76 DNA Artificial clone of aptamer 98 gggagaggag agaacgttct
cgtctgcaag gaacacaata gttagcattg cgggatcgtt 60 acgactagca tcgatg 76
99 76 DNA Artificial clone of aptamer 99 gggagaggag agaacgttct
cgcgccaaca aagctggagt acttagagcg cgggatcgtt 60 acgactagca tcgatg 76
100 76 DNA Artificial clone of aptamer 100 gggagaggag agaacgttct
cgattgcaaa atagctgtag aactaagcaa tcggatcgtt 60 acgactagca tcgatg 76
101 76 DNA Artificial clone of aptamer 101 gggagaggag agaacgttct
cgtgagatga ctatgttaag atgacgctgt tgggatcgtt 60 acgactagca tcgatg 76
102 76 DNA Artificial clone of aptamer 102 gggagaggag agaacgttct
cggganacaa ggaacncaat atttagtgaa cgggatcgtt 60 acgactagca tcgatg 76
103 76 DNA Artificial clone of aptamer 103 gggagaggag agaacgttct
cgccaaggaa cacaatagtt aggtgagaat cgggatcgtt 60 acgactagca tcgatg 76
104 75 DNA Artificial clone of aptamer 104 gggagaggag agaacgttct
cggtacaagg aacacaatag ttagtgccgt gggatcgtta 60 cgactagcat cgatg 75
105 77 DNA Artificial clone of aptamer 105 gggagaggag agaacgttct
cgattcaacg gtccaaaaaa gctgtagtac ttaggatcgt 60 tacgactagc atcgatg
77 106 76 DNA Artificial clone of aptamer 106 gggagaggag agaacgttct
cgcaatgcaa ggaacacaat agttagcagc cgggatcgtt 60 acgactagca tcgatg 76
107 76 DNA Artificial clone of aptamer 107 gggagaggag agaacgttct
cgaaaggaga aagctgaagt acttactatg cgggatcgtt 60 acgactagca tcgatg 76
108 76 DNA Artificial clone of aptamer 108 gggagaggag agaacgttct
cgcacaagga acacaatagt tagtgcaaga cgggatcgtt 60 acgactagca tcgatg 76
109 76 DNA Artificial clone of aptamer 109 gggagaggag agaacgttct
cgcacaagga actacgagtt agtgtgggag tgggatcgtt 60 acgactagca tcgatg 76
110 76 DNA Artificial clone of aptamer 110 gggagaggag agaacgttct
cgcacaagga acacaatagt tagtgcaaga cgggatcgtt 60 acgactagca tcgata 76
111 75 DNA Artificial clone of aptamer 111 gggagaggag agaacgttct
cggcgggaaa atagctgtag tactaaccca cggatcgtta 60 cgactagcat cgatg 75
112 76 DNA Artificial clone of aptamer 112 gggagaggag agaacgttct
cggcctcaag gaaaagaaaa tttagaggcc cgggatcgtt 60 acgactagca tcgatg 76
113 76 DNA Artificial clone of aptamer 113 gggagaggag agaacgttct
cggaacaaga tagctgaagg actaagttta cgggatcgtt 60 acgactagca tcgatg 76
114 76 DNA Artificial clone of aptamer 114 gggagaggag agaacgttct
cggaacaaga tagctgaagg actaagttta cgggatcgtt 60 acgactagca tcgatg 76
115 77 DNA Artificial clone of aptamer 115 gggagaggag agaacgttct
cggagccaag gaaacgaaga tttaggctca ttgggatcgt 60 tacgactagc atcgatg
77 116 76 DNA Artificial clone of aptamer 116 gggagaggag agaacgttct
cgatcacaag aaatgtggga nggtagtgat ncnnntcgtt 60 ncgactagca tcgatg 76
117 76 DNA Artificial clone of aptamer 117 gggagaggag agaacgttct
cgtcgaaagg gagctttgtc tcgggacaga acggatcgtt 60 acgactagca tcgatg 76
118 76 DNA Artificial clone of aptamer 118 gggagaggag agaacgntct
cgtgcaaaga tagctggagg actaatgcgg cgggatcgtt 60 acgactagca tcgatg 76
119 76 DNA Artificial clone of aptamer 119 gggagaggag agaacgttct
cgtcgaaagg gagctttgtc tcgggacaga acggatcgtt 60 acgactagca tcgatg 76
120 76 DNA Artificial clone of aptamer 120 gggagaggag agaacgttct
cgncnaaggn gagctttgtc ccnggacana angnatcgtt 60 acaactagca tcgatg 76
121 76 DNA Artificial clone of aptamer 121 gggagaggag agaacgttct
cggaacaaga tagctgaagg actaagttta cgggatcgtt 60 acgactagca tcgatg 76
122 76 DNA Artificial clone of aptamer 122 gggagaggag agaacgttct
cggaacaaga tagctgaagg actaagttta cgggatcgtt 60 acgactagca tcgatg 76
123 78 DNA Artificial clone of aptamer 123 gggagaggag agaacgttct
cggcgcaaaa aaagctggag tacttagtgt cgagggatcg 60 ttacgactag catcgatg
78 124 76 DNA Artificial clone of aptamer 124 gggagaggag agaacgttct
cgtcgaaagg gagctttgtc tcgggacaga acggatcgtt 60 acgactagca tcgatg 76
125 76 DNA Artificial clone of aptamer 125 gggagaggag agaacgttct
cgacacaaga aagctgcaga acttagggtc gtggatcgtt 60 acgactagca tcgatg 76
126 76 DNA Artificial clone of aptamer 126 gggagaggag agaacgttct
cggaacngga ttgttgaagg actaanttta cgggatcgtt 60 acgactagca tcgatg 76
127 76 DNA Artificial clone of aptamer 127 gggagaggag agaacgttct
cggcctcaag ggaaagaaaa tttagaggcc cgggatcgtt 60 acgactagca tcgatg 76
128 77 DNA Artificial clone of aptamer 128 gggagaggag agaacgttct
cggaaacaag cttagaaatt cgcacccttg ccgggatcgt 60 tacgactagc atcgatg
77 129 75 DNA Artificial clone of aptamer 129 gggagaggag agaacgttct
cgaaagaaaa aagctggaga acttacttcc gggatcgtta 60 cgactagcat cgatg 75
130 78 DNA Artificial clone of aptamer 130 gggagaggag agaacgttct
cggtgattgt actcacatag aaatggcaac actgggatcg 60 ttacgactag catcgatg
78 131 76 DNA Artificial clone of aptamer 131 gggagaggag agaacgttct
cgggttcaag gaacatgata gttagaaccc gcggatcgtt 60 acgactagca tcgatg 76
132 77 DNA Artificial clone of aptamer 132 gggagaggag agaacgttct
cgttccgaaa ggaacacaat agttatcgga ttgggatcgt 60 tacgactagc atcgatg
77 133 76 DNA Artificial clone of aptamer 133 gggagaggag agaacgttct
cgtctgcaag gaacacaata gttagcattg cgggatcgtt 60 acgactagca tcgatg 76
134 74 DNA Artificial clone of aptamer 134 gggagaggag agaacgttct
cggtacaagg aacacaatag ttagtgccgg ggatcgttac 60 gactagcatc gatg 74
135 75 DNA Artificial clone of aptamer 135 gggagaggag agaacgttct
cggaactcag agatcctatg tggaccagag aggatcgtta 60 cgactagcat cgatg 75
136 76 DNA Artificial clone of aptamer 136 gggagaggag agaacgttct
cgctgagcaa ggaacgtaat agttagcctg cgggatcgtt 60 acgactagca tcgatg 76
137 77 DNA Artificial clone of aptamer 137 gggagaggag agaacgttct
cgnannnata aatgatggat cncttattgt nnaggatcgt 60 tacgactagc atcgatg
77 138 74 DNA Artificial clone of aptamer 138 gggagaggag agaacgttct
cggcttggaa aaatagcttt tgggcatccg ggatcgttac 60 gactagcatc gatg 74
139 76 DNA Artificial clone of aptamer 139 gggagaggag agaacgttct
cgggttcaag gaacatgata gctagaaccc gcggatcgtt 60 acgactagca tcgatg 76
140 76 DNA Artificial clone of aptamer 140 gggagaggag agaacgttct
cgggttcaag gaacatgata gttagaaccc gcggatcgtt 60 acgactagca tcgatg 76
141 76 DNA Artificial clone of aptamer 141 gggagaggag agaacgttct
cgtgggcagg gaacacaata gttagcctac gcggatcgtt 60 acgactagca tcgatg 76
142 75 DNA Artificial clone of aptamer 142 gggagaggag agaacgttct
cgcgtgaaag gaacacaata gttatcgtgc gggatcgtta 60 cgactagcat cgatg 75
143 77 DNA Artificial clone of aptamer 143 gggagaggag agaacgttct
cgcgaggttt atcctagacg actaaccgcc tggggatcgt 60 tacgactagc atcgatg
77 144 76 DNA Artificial clone of aptamer 144 gggagaggag agaacgttct
cgtctgctag gaacacaata gttagcattg cgggatcgtt 60 acgactagca tcgatg 76
145 77 DNA Artificial clone of aptamer 145 gggagaggag agaacgttct
cgcacaagga actacgagtt agtgtgggag tggggatcgt 60 tacgactagc atcgatg
77 146 77 DNA Artificial clone of aptamer 146 gggagaggag agaacgttct
cgtgacacga ggaacttaga gttagtagca cgaggatcgt 60 tacgactagc atcgatg
77 147 76 DNA Artificial clone of aptamer 147 gggagaggag agaacgttct
cggcggcgaa ggaacacaat agttacgtcc cgggatcgtt 60 acgactagca tcgatg 76
148 76 DNA Artificial clone of aptamer 148 gggagaggag agaacgttct
cgagcccaaa aaagctgaag tactttgggc agggatcgtt 60 acgactagca tcgatg 76
149 75 DNA Artificial clone of aptamer 149 gggagaggag agaacgttct
cggtacaagg aacacaatag ttagtgccgt gggatcgtta 60 cgactagcat cgatg 75
150 45 DNA Artificial clone of aptamer 150 gggagaggag agaacgttct
cggatcgtta cgactagcat cgatg 45 151 76 DNA Artificial clone of
aptamer 151 gggagaggag agaacgttct cgtgcgcaag gaacacaata gttagggcgc
gaggatcgtt 60 acgactagca ttgatg 76 152 76 DNA Artificial clone of
aptamer 152 gggagaggag agaacgttct cggaatggaa ggaacacaat agttaccaga
cgggatcgtt 60 acgactagca tcgatg 76 153 76 DNA Artificial clone of
aptamer 153 gggagaggag agaacgttct cgtctgcaag gaacacaata gttagcattg
cgggatcgtt 60 acgactagca tcgatg 76 154 76 DNA Artificial clone of
aptamer 154 gggagaggag agaacgttct cgagacaaga cagctggagg actaagtcac
gaggatcgtt 60 acgactagca tcgatg 76 155 76 DNA Artificial clone of
aptamer 155 gggagaggag agaacgttct cgatgcccgc aaaggaacac gatagttatg
cgggatcgtt 60 acgactagca tcgatg 76 156 76 DNA Artificial clone of
aptamer 156 gggagaggag agaacgttct cgtctgnnag gaacacaata tttagcattg
cgggatcgtt 60 acgactagca tcgatg 76 157 76 DNA Artificial clone of
aptamer 157 gggagaggag agaacgttct cgaatgtgcg gagcagtatt ggtacacttt
cgggatcgtt 60 acgactagca tcgatg 76 158 76 DNA Artificial clone of
aptamer 158 gggagaggag agaacgttct cgccaaggaa cacaatagtt aggtgagaat
cgggatcgtt 60 acgactagca tcgatg 76 159 76 DNA Artificial clone of
aptamer 159 gggagaggag agaacgttct cgccaaggaa cacaatagtt aggtgagaat
cgggatcgtt 60 acgactagca tcgatg 76 160 76 DNA Artificial clone of
aptamer 160 gggagaggag agaacgttct cgggaagcaa ggaacttaga gttagttgac
cgggatcgtt 60 acgactagca tcgatg 76 161 76 DNA Artificial clone of
aptamer 161 gggagaggag agaacgttct cgtgggcaag gaacacaata gttagcctac
gcggatcgtt 60 acgactagca tcgatg 76 162 76 DNA Artificial clone of
aptamer 162 gggagaggag agaacgttct cgtcgggcat ggaacacaat agttagaccg
cgggatcgtt 60 acgactagca tcgatg 76 163 75 DNA Artificial clone of
aptamer 163 gggagaggag agaacgttct cggtcgcaag gaacataata gttagcggag
gggatcgtta 60 cgactagcat cgatg 75 164 76 DNA Artificial clone of
aptamer 164 gggagaggag agaacgttct cgtctgcaag gaacacaata gttagcattg
cgggatcgtt 60 acgactagca tcgatg 76 165 77 DNA Artificial clone of
aptamer 165 gggagaggag agaacgttct cgccgacaat cagctcggat cgtgtgctac
gctggatcgt 60 tacgactagc atcgatg 77 166 77 DNA Artificial clone of
aptamer 166 gggagaggag agaacgttct cgagacaaga tagctgaagg actaagtcac
gagggatcgt 60 tacgactagc atcgatg 77 167 76 DNA Artificial clone of
aptamer 167 gggagaggag agaacgttct cggaacaaga tagctgaagg actaagtttg
cgggatcgtt 60 acgactagca tcgatg 76 168 77 DNA Artificial clone of
aptamer 168 gggagaggag agaacgttct cggagncaag gaaacnaata tttaggctca
ntggnnncnt 60 tncanctagc nncnnta 77 169 76 DNA Artificial clone of
aptamer 169 gggagaggag agaacgttct cgtctgcaag gaacacaata gttagcattg
cgggatcgtt 60 acgactagca tcgatg 76 170 76 DNA Artificial clone of
aptamer 170 gggagaggag agaacgttct cggaacaaga tagctgaagg actaagttta
cgggatcgtt 60 acgactagca tcgatg 76 171 45 DNA Artificial clone of
aptamer 171 gggagaggag agaacgttct cggatcgtta cgactagcat cgatg 45
172 78 DNA Artificial clone of aptamer 172 gggagaggag agaacgttct
cggtgatagt actcacatag aaatggctac actgggatcg 60 ttacgactag catcgatg
78 173 76 DNA Artificial clone of aptamer 173 gggagaggag agaacgttct
cgcctgggca aggaacagaa aagttagcgc caggatcgtt 60 acgactagca tcgatg 76
174 76 DNA Artificial clone of aptamer 174 gggagaggag agaacgttct
cgtaacggac aaaaggaacc gggaagttat ctggatcgtt 60 acgactagca tcgatg 76
175 76 DNA Artificial clone of aptamer 175 gggagaggag agaacgttct
cgcgcacaag atagagaaga ctaagtccgc ggggatcgtt 60 acgactagca tcgatg 76
176 76 DNA Artificial clone of aptamer 176 gggagaggag agaacgttct
cgcgcacaag atagagaaga ctaagttcgc ggggatcgtt 60 acgactagca tcgatg 76
177 76 DNA Artificial clone of aptamer 177 gggagaggag agaacgttct
cgcgccaata aagctggagt acttagagcg cgggatcgtt 60 acgactagca tcgatg 76
178 76 DNA Artificial clone of aptamer 178 gggagaggag agaacgttct
cgggaaacaa ggaacttaga gttagttgac cgggatcgtt 60 acgactagca tcgatg 76
179 76 DNA Artificial clone of aptamer 179 gggagaggag agaacgttct
cgctagcaag ataggtggga ctaagctagt gaggatcgtt 60 acgactagca tcgatg 76
180 76 DNA Artificial clone of aptamer 180 gggagaggag agaacgttct
cgtcgaaggg gagctttgtc tcgggacaga acggatcgtt 60 acgactagca tcgatg 76
181 76 DNA Artificial clone of aptamer 181 gggagaggag agaacgttct
cggaacaaga tagctgaagg actaagttta cgggatcgtt 60 acgactagca tcgatg 76
182 76 DNA Artificial clone of aptamer 182 gggagaggag agaacgttct
cggaacaaga tagctgaagg actaagtttg cgggatcgtt 60 acgactagca tcgatg 76
183 77 DNA Artificial clone of aptamer 183 gggagaggag anntccccnc
ncggaaaaan aaaaaagaag aantangttn gggggatcgt 60 tacgactagc atcgatg
77 184 30 RNA Artificial r/mGmH aptamer ARC224 -Stabilized VEGF
Aptamer 184 cgnunugcng uuugngnngu cgcgcnuucg 30 185 30 RNA
Artificial r/mGmH aptamer ARC225 - Stabilized VEGF Aptamer 185
cgnunugcng uuugngnngu cgcgcnuucg 30 186 24 RNA Artificial r/mGmH
aptamer ARC226 Single-hydroxy VEGF aptamer 186 gnucnugcnu
guggnucgcg gnuc 24 187 23 RNA Artificial r/mGmH aptamer ARC245 VEGF
Aptamer 187 nugcnguuug ngnngucgcg cnu 23 188 23 RNA Artificial
r/mGmH aptamer ARC259 hVEGF Aptamer 188 ncgcnguuug ngnngucgcg cgu
23 189 82 DNA Artificial clone of aptamer 189 gggagaggag agaacgttct
actatgaagg gttttaaaga tgacacatta gccgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 190 75 DNA Artificial clone of aptamer 190
gggagaggag agaacgttct acaggcagtt ctggggaccc atgggggaag tgcgctgtcg
60 atcgatcgat cgatg 75 191 75 DNA Artificial clone of aptamer 191
gggagaggag agaacgttct acgattagca gggagggaga gtgcgaagag gacgctgtcg
60 atcgatcgat cgatg
75 192 75 DNA Artificial clone of aptamer 192 gggagaggag agaacgttct
acactctggg gacccgtggg ggagtgcagc aacgctgtcg 60 atcgatcgat cgatg 75
193 75 DNA Artificial clone of aptamer 193 gggagaggag agaacgttct
acaagcagtt ctggggaccc atgggggaag tgcgctgtcg 60 atcgatcgat cgatg 75
194 74 DNA Artificial clone of aptamer 194 gggagaggag agaacgttct
acgaggtgag ggtctacaat ggagggatgg tcgctgtcga 60 tcgatcgatc gatg 74
195 75 DNA Artificial clone of aptamer 195 gggagaggag agaacgttct
acccgcagca tagcctgngg acccatgngg ggcgctgtcg 60 atcgatcgat cgatg 75
196 75 DNA Artificial clone of aptamer 196 gggagaggag agaacgttct
actggggggc gtgttcatta gcagcgtcgt gtcgctgtcg 60 atcgatcgat cgatg 75
197 75 DNA Artificial clone of aptamer 197 gggagaggag agaacgttct
acaggcagtt ctggggaccc atgggggaag tgcgctgtcg 60 atcgatcgat cgatg 75
198 75 DNA Artificial clone of aptamer 198 gggagaggag agaacgttct
acgcagcgca tctggggacc caagagggga ttcgctgtcg 60 atcgatcgat cgatg 75
199 75 DNA Artificial clone of aptamer 199 gggagaggag agaacgttct
acaggcagtt ctggggaccc atgggggaag tgcgctgtcg 60 atcgatcgat cgatg 75
200 73 DNA Artificial clone of aptamer 200 gggagaggag agaacgttct
acgggatggg tagttggatg gaaatgggaa cgctgtcgat 60 cgatcgatcg atg 73
201 74 DNA Artificial clone of aptamer 201 gggagaggag agaacgttct
acgaggtgta gggatagagg ggtgtaggta acgctgtcga 60 tcgatcgatc gatg 74
202 75 DNA Artificial clone of aptamer 202 gggagaggag agaacgttct
acaggagtgg agctacagag agggttaggg gtcgctgtcg 60 atcgatcgat cgatg 75
203 75 DNA Artificial clone of aptamer 203 gggagaggag agaacgttct
acggatgttg ggagtgatag aaggaagggg agcgctgtcg 60 atcgatcgat cgatg 75
204 75 DNA Artificial clone of aptamer 204 gggagaggag agaacgttct
acaggcagtt ctggggaccc atgggggaag tgcgctgtcg 60 atcgatcgat cgatg 75
205 75 DNA Artificial clone of aptamer 205 gggagaggag agaacgttct
acaggcagtt ctggggaccc atgggggaag tgcgctgtcg 60 atcgatcgat cgatg 75
206 75 DNA Artificial clone of aptamer 206 gggagaggag agaacgttct
acaggcagtt ctggggaccc atgggggaag tgcgctgtcg 60 atcgatcgat cgatg 75
207 76 DNA Artificial clone of aptamer 207 gggagaggag agaacgttct
acttggggtg gaaggagtaa gggaggtgct gatcgctgtc 60 gatcgatcga tcgatg 76
208 75 DNA Artificial clone of aptamer 208 gggagaggag agaacgttct
acgtattagg ggggaagggg aggaatagat cacgctgtcg 60 atcgatcgat cgatg 75
209 76 DNA Artificial clone of aptamer 209 gggagaggag agaacgttct
acagggagag agtgttgagt gaagaggagg agtcgctgtc 60 gatcgatcga tcgatg 76
210 75 DNA Artificial clone of aptamer 210 gggagaggag agaacgttct
acattgtgct cctggggccc agtggggagc cacgctgtcg 60 atcgatcgat cgatg 75
211 75 DNA Artificial clone of aptamer 211 gggagaggag agaacgttct
acgagcagcc ctggggcccg gagggggatg gtcgctgtcg 60 atcgatcgat cgatg 75
212 75 DNA Artificial clone of aptamer 212 gggagaggag agaacgttct
acaggcagtt ctggggaccc atgggggaag tgcgctgtcg 60 atcgatcgat cgatg 75
213 75 DNA Artificial clone of aptamer 213 gggagaggag agaacgttct
accaacggca tcctgggccc cacaggggat gtcgctgtcg 60 atcgatcgat cgatg 75
214 74 DNA Artificial clone of aptamer 214 gggagaggag agaacgttct
acgagtggat agggaagaag gggagtagtc acgctgtcga 60 tcgatcgatc gatg 74
215 75 DNA Artificial clone of aptamer 215 gggagaggag agaacgttct
acccgcagca tagcctgggg acccatgggg ggcgctgtcg 60 atcgatcgat cgatg 75
216 76 DNA Artificial clone of aptamer 216 gggagaggag agaacgttct
acggtcgcgt gtgggggacg gatgggtatt ggtcgctgtc 60 natcgatcga tcnatg 76
217 75 DNA Artificial clone of aptamer 217 gggagaggag agaacgttct
acccgcagca tagcctgggg acccatgggg ggcgctgtcg 60 atcgatcgat cgatg 75
218 75 DNA Artificial clone of aptamer 218 gggagaggag agaacgttct
acccgcagca tagcctgggg acccatgggg ggcgctgtcg 60 atcgatcgat cgatg 75
219 75 DNA Artificial clone of aptamer 219 gggagaggag agaacgttct
acggggttac gtcgcacgat acatgcattc atcgctgtcg 60 atcgatcgat cgatg 75
220 75 DNA Artificial clone of aptamer 220 gggagaggag agaacgttct
actagcgagg aggggttttc tatttttgcg atcgctgtcg 60 atcgatcgat cgatg 75
221 75 DNA Artificial clone of aptamer 221 gggagaggag agaacgttct
acgtgtgatg gggtgagagg atgagttagt gacgctgtcg 60 atcgatcgat cgatg 75
222 74 DNA Artificial clone of aptamer 222 gggagaggag agaacgttct
acaatgggag ggtaatagtg atgaggagag gcgctgtcga 60 tcgatcgatc gatg 74
223 75 DNA Artificial clone of aptamer 223 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
224 75 DNA Artificial clone of aptamer 224 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
225 75 DNA Artificial clone of aptamer 225 gggagaggag agaacgttct
acaggtagcg tgagggggtg ttaatagagg ggcgctgtcg 60 atcgatcgat cgatg 75
226 75 DNA Artificial clone of aptamer 226 gggagaggag agaacgttct
acgataggat gggtgggaca ggagagggag tgcgctgtcg 60 atcgatcgat cgatg 75
227 75 DNA Artificial clone of aptamer 227 gggagaggag agaacgttct
accagtgagg gcagtgtcag attgagagga ggcgctgtcg 60 atcgatcgat cgatg 75
228 75 DNA Artificial clone of aptamer 228 gggagaggag agaacgttct
accttgccta acaggaggtg gagtattgga cccgctgtcg 60 atcgatcgat cgatg 75
229 75 DNA Artificial clone of aptamer 229 gggagaggag agaacgttct
accttgccta acaggaggtg gagtattgga cccgctgtcg 60 atcgatcgat cgatg 75
230 73 DNA Artificial clone of aptamer 230 gggagaggag agaacgttct
acgtcgtgag taatggctcg tagatgaggt cgctgtcgat 60 cgatcgatcg atg 73
231 74 DNA Artificial clone of aptamer 231 gggagaggag agaacgttct
acgggattaa gaggggagag gagcagttga gcgctgtcga 60 tcgatcgatc gatg 74
232 75 DNA Artificial clone of aptamer 232 gggagaggag agaacgttct
actccggttg gggtatcagg tctacggact gacgctgtcg 60 atcgatcgat cgatg 75
233 75 DNA Artificial clone of aptamer 233 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
234 75 DNA Artificial clone of aptamer 234 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
235 75 DNA Artificial clone of aptamer 235 gggagaggag agaacgttct
acatgacaag agggggttgt gtgggatggc agcgctgtcg 60 atcgatcgat cgatg 75
236 76 DNA Artificial clone of aptamer 236 gggagaggag agaacgttct
acacagggag gggagcggag aggagagagg gtacgctgtc 60 gatcgatcga tcgatg 76
237 75 DNA Artificial clone of aptamer 237 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
238 73 DNA Artificial clone of aptamer 238 gggagaggag agaacgttct
acgtcgtgag taatggctcg tagatgaggt cgctgtcgat 60 cgatcgatcg atg 73
239 75 DNA Artificial clone of aptamer 239 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
240 75 DNA Artificial clone of aptamer 240 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
241 75 DNA Artificial clone of aptamer 241 gggagaggag agaacgttct
accttgccta acaggaggtg gagtattgga cccgctgtcg 60 atcgatcgat cgatg 75
242 75 DNA Artificial clone of aptamer 242 gggagaggag agaacgttct
acggctatgc gtcgtgagtc aatggcccgc atcgctgtcg 60 atcgatcgat cgatg 75
243 75 DNA Artificial clone of aptamer 243 gggagaggag agaacgttct
acgggtcgtg agatagtggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
244 75 DNA Artificial clone of aptamer 244 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
245 75 DNA Artificial clone of aptamer 245 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
246 75 DNA Artificial clone of aptamer 246 gggagaggag agaacgttct
accttgtcta acaggaggtg gagtattgga cccgctgtcg 60 atcgatcgat cgatg 75
247 75 DNA Artificial clone of aptamer 247 gggagaggag agaacgttct
acgactttga gggtggtgag agtggaagag agcgctgtcg 60 atcgatcgat cgatg 75
248 75 DNA Artificial clone of aptamer 248 gggagaggag agaacgttct
acggtagggt atgaccaggg aggtattgga ggcgctgtcg 60 atcgatcgat cgatg 75
249 75 DNA Artificial clone of aptamer 249 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
250 75 DNA Artificial clone of aptamer 250 gggagaggag agaacgttct
acgggtcgtg agataatggc tcccgtattc agcgctgtcg 60 atcgatcgat cgatg 75
251 72 DNA Artificial clone of aptamer 251 gggagaggag agaacgttct
acgttatgca tgtggagagt gagagagggc gctgtcgatc 60 gatcgatcga tg 72 252
75 DNA Artificial clone of aptamer 252 gggagaggag agaacgttct
accatgtctg cgggaggtga gtagtgatcc tgcgctgtcg 60 atcgatcgat cgatg 75
253 75 DNA Artificial clone of aptamer 253 gggagaggag agaacgttct
acagagtggg agggatgtgt gacacaggta ggcgctgtcg 60 atcgatcgat cgatg 75
254 73 DNA Artificial clone of aptamer 254 gggagaggag agaacgttct
acgctccatg acagtgaggt gagtagtgat cgctgtcgat 60 cgatcgatcg atg 73
255 74 DNA Artificial clone of aptamer 255 gggagaggag agaacgttct
cgatgctgac agggtgtgtt cagtaatggc tcgctgtcga 60 tcgatcgatc gatg 74
256 75 DNA Artificial clone of aptamer 256 gggagaggag agaacgttct
accagcaaac agggtcaggt gagtagtgat gacgctgtcg 60 atcgatcgat cgatg 75
257 75 DNA Artificial clone of aptamer 257 gggagaggag agaacgttct
acgacaagcc gggggtgttc agtagtggca accgctgtcg 60 atcgatcgat cgatg 75
258 75 DNA Artificial clone of aptamer 258 gggagaggag agaacgttct
acatatggcg ctggaggtga gtaatgatcg tgcgctgtcg 60 atcgatcgat cgatg 75
259 75 DNA Artificial clone of aptamer 259 gggagaggag agaacgttct
acggggcgat agcgttcagt agtggcgccg gtcgctgtcg 60 atcgatcgat cgatg 75
260 74 DNA Artificial clone of aptamer 260 gggagaggag agaacgttct
acatagcgga ctgggtgcat ggagcggcgc acgctgtcga 60 tcgatcgatc gatg 74
261 74 DNA Artificial clone of aptamer 261 gggagaggag agaacgttct
acgggtcaac aggggcgttc agtagtggcg gcgctgtcga 60 tcgatcgatc gatg 74
262 75 DNA Artificial clone of aptamer 262 gggagaggag agaacgttct
acgcatgcga gctgaggtga gtagtgatca gtcgctgtcg 60 atcgatcgat cgatg 75
263 74 DNA Artificial clone of aptamer 263 gggagaggag agaacgttct
acatgcgaca ggggagtgtt cagtagtggc acgctgtcga 60 tcgatcgatc gatg 74
264 75 DNA Artificial clone of aptamer 264 gggagaggag agaacgttct
accccatcgt atggagtgcg gaacggggca tacgctgtcg 60 atcgatcgat cgatg 75
265 72 DNA Artificial clone of aptamer 265 gggagaggag agaacgttct
acagtgaggc gggagcgttt cagtaatggc gctgtcgatc 60 gatcgatcga tg 72 266
74 DNA Artificial clone of aptamer 266 gggagaggag agaacgttct
acacagcgtc gggtgttcag taatggcgca gcgctgtcga 60 tcgatcgatc gatg 74
267 75 DNA Artificial clone of aptamer 267 gggagaggag agaacgttct
acggtgttca gtagtggcac aggaggaagg gatgctgtcg 60 atcgatcgat cgatg 75
268 75 DNA Artificial clone of aptamer 268 gggagaggag agaacgttct
acagttcagg cgttaggcat gggtgtcgct ttcgctgtcg 60 atcgatcgat cgatg 75
269 75 DNA Artificial clone of aptamer 269 gggagaggag agaacgttct
acatgcgaca tgcgagtgtt cagtagcggc agcgctgtcg 60 atcgatcgat cgatg 75
270 75 DNA Artificial clone of aptamer 270 gggagaggag agaacgttct
acctatggcg ttacagcgag gtgagtagtg atcgctgtcg 60 atcgatcgat cgatg 75
271 75 DNA Artificial clone of aptamer 271 gggagaggag agaacgttct
accagccgat ccagccaggc gttcagtagt ggcgctgtcg 60 atcgatcgat cgatg 75
272 74 DNA Artificial clone of aptamer 272 gggagaggag agaacgttct
acggcacagg cacggcgagg tgagtaatga tcgctgtcga 60 tcgatcgatc gatg 74
273 73 DNA Artificial clone of aptamer 273 gggagaggag agaacgttct
actgtggaca gcgggagtgc ggaacggggt cgctgtcgat 60 cgatcgatcg atg 73
274 75 DNA Artificial clone of aptamer 274 gggagaggag agaacgttct
actgatgctg cgagtgcatg gggcaggcgc ttcgctgtcg 60 atcgatcgat cgatg 75
275 75 DNA Artificial clone of aptamer 275 gggagaggag agaacgttct
acggtacaat gggaatgaca gtgatgggta gccgctgtcg 60 atcgatcgat cgatg 75
276 73 DNA Artificial clone of aptamer 276 gggagaggag agaacgttct
acatggacag cgaagcatgg gggaggcgca cgctgtcgat 60 cgatcgatcg atg 73
277 75 DNA Artificial clone of aptamer 277 gggagaggag agaacgttct
actgggagcg acagtgagca tggggtaggc gccgctgtcg 60 atcgatcgat cgatg 75
278 74 DNA Artificial clone of aptamer 278 gggagaggag agaacgttct
accggcgagc aggtgttcag tagtggcttt gcgctgtcga 60 tcgatcgatc gatg 74
279 75 DNA Artificial clone of aptamer 279 gggagaggag agaacgttct
acgatcagtg agggagtgca gtagtggctc gtcgctgtcg 60 atcgatcgat cgatg 75
280 81 DNA Artificial clone of aptamer 280 gggagaggag agaacgttct
acaaatgaga gcaggccgaa aaggagtcgc tcgctgtcga 60 tcgatcgatc
gatgaagggc g 81 281 82 DNA Artificial clone of aptamer 281
gggagaggag agaacgttct acaaaggatc aatctttcgg cgtatgtgtg agcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 282 82 DNA Artificial clone of
aptamer 282 gggagaggag agaacgttct acggtaaagc aggctgactg aaaggttgaa
gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 283 81 DNA Artificial
clone of aptamer 283 gggagaggag agaacgttct acaggttaaa agcaggctca
ggaatggaag tcgctgtcga 60 tcgatcgatc gatgaagggc g 81 284 82 DNA
Artificial clone of aptamer 284 gggagaggag agaacgttct acaacaaagc
aggctcatag taatatggaa gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 285
81 DNA Artificial clone of aptamer 285 gggagaggag agaacgttct
acaaaagaga gcaggccgaa aaggagtcgc tcgctgtcga 60 tcgatcgatc
gatgaagggc g 81
286 82 DNA Artificial clone of aptamer 286 gggagaggag agaacgttct
acaaaaggca ggctcagggg atcactggaa gtcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 287 82 DNA Artificial clone of aptamer 287
gggagaggag agaacgttct acaaaaagca ggccgtatgg atataaggga gtcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 288 82 DNA Artificial clone of
aptamer 288 gggagaggag agaacgttct acaaaagtgc aggctgcaga catatgcgaa
gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 289 81 DNA Artificial
clone of aptamer 289 gggagaggag agaacgttct acaaaggaga gcaggccgaa
aaggagtcgc tcgctgtcga 60 tcgatcgatc gatgaagggc g 81 290 83 DNA
Artificial clone of aptamer 290 gggagaggag agaacgttct acaagatata
attaaggata agtgcaaagg agacgctgtc 60 gatcgatcga tcgatgaagg gcg 83
291 84 DNA Artificial clone of aptamer 291 gggagaggag agaacgttct
acagacaaca gcnagaggga atcncanaca aagacgctgt 60 cgatcgatcg
atcgatgaag ggcg 84 292 82 DNA Artificial clone of aptamer 292
gggagaggag agaacgttct acagattcta agcgcaggaa taagtcacca gacgctgtcg
60 atcgatcgat cgatgaaggg cg 82 293 82 DNA Artificial clone of
aptamer 293 gggagaggag agaacgttct acgaaaatga gcatggaagt gggagtacgt
gccgctgtcg 60 atcgatcgat cgatgaaggg cg 82 294 82 DNA Artificial
clone of aptamer 294 gggagaggag agaacgttct acgaaaagag gcgccggaag
tgagagtaag tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 295 82 DNA
Artificial clone of aptamer 295 gggagaggag agaacgttct acgaagtgag
tttccgaagt gagagtacga aacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 296
81 DNA Artificial clone of aptamer 296 gggagaggag agaacgttct
acgaatgaga gcaggccgaa aaggagtcgc tcgctgtcga 60 tcgatcgatc
gatgaagggc g 81 297 82 DNA Artificial clone of aptamer 297
gggagaggag agaacgttct acgagaggca agagagagtc gcataaaaaa gacgctgtcg
60 atcgatcgat cgatgaaggg cg 82 298 82 DNA Artificial clone of
aptamer 298 gggagaggag agaacgttct acgcaggctg tcgtagacaa acgatgaagt
cgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 299 83 DNA Artificial
clone of aptamer 299 gggagaggag agaacgttct acggaaaaag atatgaaaga
aaggattaag agacgctgtc 60 gatcgatcga tcgatgaagg gcg 83 300 82 DNA
Artificial clone of aptamer 300 gggagaggag agaacgttct acggaaggna
acaanagcac tgtttgtgca ggcgctgtcg 60 atcnatcnat cnatgaaggg cg 82 301
82 DNA Artificial clone of aptamer 301 gggagaggag agaacgttct
acggagcata nggcntgaaa ctgaganagt aacgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 302 83 DNA Artificial clone of aptamer 302
gggagaggag agaacgttct acgaaaaagg atatgagaga aaggattaag agacgctgtc
60 gatcgatcga tcgatgaagg gcg 83 303 82 DNA Artificial clone of
aptamer 303 gggagaggag agaacgttct acatacatag gcgccgcgaa tgggaaagaa
agcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 304 82 DNA Artificial
clone of aptamer 304 gggagaggag agaacgttct actcatgaag ccatggttgt
aattctgttt ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 305 80 DNA
Artificial clone of aptamer 305 gggagaggag agaacgttct actaatgcag
gctcagttac tactggaagt cgctgtcgat 60 cgatcgatcg atgaagggcg 80 306 81
DNA Artificial clone of aptamer 306 gggagaggag agaacgttct
actttcatag gcgggattat ggaggagtat tcgctgtcga 60 tcgatcgatc
gatgaagggc g 81 307 82 DNA Artificial clone of aptamer 307
aggagaggag agaacgttct actagaagca ggctcgaata caattcggaa gtcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 308 82 DNA Artificial clone of
aptamer 308 gggagaggag agaacgttct acttagcgat gtcggaagag agagtacgag
gacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 309 82 DNA Artificial
clone of aptamer 309 gggagaggag agaacgttct acttgcgaag accgtggaag
aggagtactg gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 310 82 DNA
Artificial clone of aptamer 310 gggagaggag agaacgttct acttttggtg
aaggtgtaag agtggcacta cacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 311
82 DNA Artificial clone of aptamer 311 gggagaggag agaacgttct
accatcagtt gtggcgatta tgtgggagta tgcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 312 83 DNA Artificial clone of aptamer 312
gggagaggag agaacgttct acanaanaac atgcgattaa agatcatgaa cagcgctgtc
60 gatcgatcga tcgatgaagg gcg 83 313 82 DNA Artificial clone of
aptamer 313 gggagaggag agaacgttct acataagcag gctccgatag tattcgggaa
gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 314 82 DNA Artificial
clone of aptamer 314 gggagaggag agaacgttct actttcggaa tgcgatgggg
gtgattcgtg gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 315 80 DNA
Artificial clone of aptamer 315 gggagaggag agaacgttct acctgttgag
gctaagtgga tgattgaggg cgctgtcgat 60 cgatcgatcg atgaagggcg 80 316 81
DNA Artificial clone of aptamer 316 gggagaggag agaacgttct
acctgggtcg gtgcgattgg agatgtcgtt gcgctgtcga 60 tcgatcgatc
gatgaagggc g 81 317 82 DNA Artificial clone of aptamer 317
gggagaggag agaacgttct acctgatgtc aggttgtttg gagattatct gacnctgtcn
60 atcgatcgat cgatgaaggg cg 82 318 82 DNA Artificial clone of
aptamer 318 gggagaggag agaacgttct acctcgcgcg acgagcgaat ttccggatgc
ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 319 82 DNA Artificial
clone of aptamer 319 gggagaggag agaacgttct accatgaatg attgcgatcg
ttgttcgtgt ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 320 83 DNA
Artificial clone of aptamer 320 gggagaggag agaacgttct actccgacca
cgcctgggtg attcctacna cgacgctgtc 60 gatcgatcga tcgatgaagg gcg 83
321 82 DNA Artificial clone of aptamer 321 gggagaggag agaacgttct
actacttttg gggattcact ccgcgctgat gtcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 322 82 DNA Artificial clone of aptamer 322
gggagaggag anaacgttct antagtgctt gcgagatagt gtaggattat actgctgtcg
60 atcgatcgat cgatgaaggg cg 82 323 82 DNA Artificial clone of
aptamer 323 gggagaggag agaacgttct actagtgtcc ttctccacgt ggttgtaatt
gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 324 82 DNA Artificial
clone of aptamer 324 gggagaggag agaacgttct actattgtgg cgcttgttgg
actaactgac tacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 325 82 DNA
Artificial clone of aptamer 325 gggagaggag agaacgtcct acttcgattg
tgatcttgtg gcggcctgtg agcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 326
82 DNA Artificial clone of aptamer 326 gggagaggag agaacgttct
acttggcgat gtcggaagag agagtacgag ggcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 327 82 DNA Artificial clone of aptamer 327
gggagaggag agaacgttct acttgaanct gcgtgaattg anagtaacga agcgctgtca
60 atcgatcnat caatnaaggg cg 82 328 82 DNA Artificial clone of
aptamer 328 gggagaggag agaacgttct actcgagagg acatgtggat ccggttcgcg
tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 329 82 DNA Artificial
clone of aptamer 329 gggagaggag agaacgttct actgtgatgc ggtttgcgtc
gaccggattc gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 330 81 DNA
Artificial clone of aptamer 330 gggagaggag agaacgttct actgtgtgat
tgggcgcatg tcgaggcgac acgctgtcga 60 tcgatcgatc gatgaagggc g 81 331
81 DNA Artificial clone of aptamer 331 gggagaggag agaacgttct
actgattaag atgcgctggt agagcggtgg gcgctgtcga 60 tcgatcgatc
gatgaagggc g 81 332 82 DNA Artificial clone of aptamer 332
gggagaggag agaacgttct actggttaat ttgcatgcgc gantaacntg ntcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 333 81 DNA Artificial clone of
aptamer 333 gggagaggag agaacgttct actgggaagc ggtaacttgg attgaccgat
ccgctgtcga 60 tcgatcgatc gatgaagggc g 81 334 82 DNA Artificial
clone of aptamer 334 gggagaggag agaacgttct actgttacgg agatgatggg
tttggctgtt ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 335 81 DNA
Artificial clone of aptamer 335 gggagaggag agaacgttct acttgtggac
tgagatacga ttcggagctg gcgctgtcga 60 tcgatcgatc gatgaagggc g 81 336
82 DNA Artificial clone of aptamer 336 gggagaggag agaacgttct
acttgtgagt ttccttgggc cttgagcgtg ggcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 337 82 DNA Artificial clone of aptamer 337
gggagaggag agaacgttct acaggtgatg tgagccgatt gtgaagtttt gtcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 338 82 DNA Artificial clone of
aptamer 338 gggagaggag agaacgttct acagcggatg tttgggggtg tgtgttggtt
gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 339 82 DNA Artificial
clone of aptamer 339 gggagaggag agaacgttct acatgcggtg gtggtcttcg
atgggtggaa gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 340 82 DNA
Artificial clone of aptamer 340 gggagaggag agaacgttct acattggagg
ggcgcatgtg gtctgtttga tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 341
82 DNA Artificial clone of aptamer 341 gggagaggag agaacgttct
acgtgtttcg cggatttgaa gaggagtaaa atcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 342 82 DNA Artificial clone of aptamer 342
gggagaggag agaacgttct acgtgtgcgt gttcgggaag ggagagtgcc gaggctgtcg
60 atcgatcgat cgatgaaggg cg 82 343 82 DNA Artificial clone of
aptamer 343 gggagaggag agaacgttct acgtgtgtgg tgtgcgatgc ttggctgttt
gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 344 82 DNA Artificial
clone of aptamer 344 gggagaggag agaacgttct acggtttgtg tggcttggat
ctgaagacta agcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 345 82 DNA
Artificial clone of aptamer 345 gggagaggag agaacgttct acggttctgg
gcttgtgtgt gaggattgac ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 346
74 DNA Artificial clone of aptamer 346 gggagaggag agaacgttct
acgatgatga aggcgaaaag acgaggctgt cgatcgatcg 60 atcgatgaag ggcg 74
347 82 DNA Artificial clone of aptamer 347 gggagaggag agaacgttct
acgagtgctg atgcgtgtcc tgggatggaa ttcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 348 82 DNA Artificial clone of aptamer 348
gggagaggag agaacgttct acgcgtttat agcgatcgat gatgatatag gccgctgtcg
60 atcgatcgat cgatgaaggg cg 82 349 82 DNA Artificial clone of
aptamer 349 gggagaggag agaacgttct acgcgttcaa atgggataga attggctgcg
ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 350 79 DNA Artificial
clone of aptamer 350 gggagaggag agaacgttct acgaaattgt gcgtcagtgt
gaggcggttt gctgtcgatc 60 gatcgatcga tgaagggcg 79 351 82 DNA
Artificial clone of aptamer 351 gggagaggag agaacgttct acggtcgaaa
tgagggtctg gagttccgac gacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 352
82 DNA Artificial clone of aptamer 352 gggagaggag agaacgttct
acgaatttgg taatctgggt gacttaggat gtcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 353 81 DNA Artificial clone of aptamer 353
gggagaggag agaacgttct acgatttttt gtgccgaagt aagagtacgc gcgctgtcga
60 tcgatcgatc gatgaagggc g 81 354 82 DNA Artificial clone of
aptamer 354 aggagaggag agaacgttct acggagtgtg cgcggatgaa aacagaagtt
gtcgctgtcn 60 atcgatcnat caatgaaggg cg 82 355 82 DNA Artificial
clone of aptamer 355 gggagaggag agaacgttct acgatctggg cgagccagtc
tgactgagga agcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 356 82 DNA
Artificial clone of aptamer 356 gggagaggag agaacgttct acgaagaaga
tatgagagaa aggattaaga gacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 357
82 DNA Artificial clone of aptamer 357 gggagaggag agaacgttct
acgaaaaaga tatgagagaa aggattaaga gacgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 358 82 DNA Artificial clone of aptamer 358
gggagaggag agaacgttct acgaaaaaga tatgagagaa aggattaaga ggcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 359 82 DNA Artificial clone of
aptamer 359 gggagaggag agaacgttct acgaaaaaga catgagagaa aggattaaga
gacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 360 83 DNA Artificial
clone of aptamer 360 gggagaggag agaacgttct acnaaaaagt atatgagaga
aaggattaan agacgctgtc 60 gatcgatcga tcgatgaagg gcg 83 361 83 DNA
Artificial clone of aptamer 361 gggagaggag agaacgttct acgaaaaaga
tatgagagaa aaggattgag agatgctgtc 60 gatcgatcga tcgatgaagg gcg 83
362 83 DNA Artificial clone of aptamer 362 gggagaggag agcacgttct
acgaaaaaga tatggagaga aaggattaag agacgctgtc 60 gatcgatcga
tcgatgaagg gcg 83 363 84 DNA Artificial clone of aptamer 363
gggagaggag agaacgttct acgaaaaaga tatgagagaa aggattaaaa gagacgctgt
60 cgatcgatcg atcgatgaag ggcg 84 364 85 DNA Artificial clone of
aptamer 364 gggagaggag agaacgttct acgaanaaga tacatagtag aaaggattaa
taagacgctg 60 tcgatcgatc gatcgatgaa gggcg 85 365 82 DNA Artificial
clone of aptamer 365 gggagaggag agaacgttct acaggcgtgt tggtagggta
cgacgaggca tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 366 82 DNA
Artificial clone of aptamer 366 gggagaggag agaacgttct acgcaaaaat
gtgatgcgag gtaatggaac gccgctgtcg 60 atcgatcgat cgatgaaggg cg 82 367
82 DNA Artificial clone of aptamer 367 gggagaggag agaacgttct
acggacctca gcgatagggg ttgaaaccga cacgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 368 82 DNA Artificial clone of aptamer 368
gggagaggag agaacgttct acatggtcgg atgctgggga gtaggcaagg ttcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 369 82 DNA Artificial clone of
aptamer 369 gggagaggag agaacgttct acgtatcggc gagcgaagca tccgggagcg
ttcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 370 82 DNA Artificial
clone of aptamer 370 gggagaggag agaacgttct acgtattggc gcgcgaagca
tccgggagcg ttcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 371 82 DNA
Artificial clone of aptamer 371 gggagaggag agaacgttct acttatacct
gacggccgga ggcgcatagg tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 372
82 DNA Artificial clone of aptamer 372 gggagaggag agaacgttct
acatggtcgg atgctgggga gtaggcaagg ttcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 373 82 DNA Artificial clone of aptamer 373
gggagaggag agaacgttct acacgagagt actgaggcgc ttggtacaga gtcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 374 82 DNA Artificial clone of
aptamer 374 gggagaggag agaacgttct acagaaggta gaaaaaggat agctgtgaga
agcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 375 82 DNA Artificial
clone of aptamer 375 gggagaggag agaacgttct actgagggat aatacgggtg
ggattgtctt cccgctgtcg 60 atcgatcgat cgatgaaggg cg 82 376 84 DNA
Artificial clone of aptamer 376 gggagaggag agaacgttct acattgagcg
ttgaagttgg ggaagctccg aggccgctgt 60 cgatcgatcg atcgatgaag ggcg 84
377 82 DNA Artificial clone of aptamer 377 gggagaggag agaacgttct
acgcggagat atacagcgag gtaatggaac gccgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 378 82 DNA Artificial clone of aptamer 378
gggagaggag agaacgttct acgaagacag cccaatagcg gcacggaact tgcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 379 84 DNA Artificial clone of
aptamer 379 gggagaggag agaacgttct accggttgag ggctcgcgtg gaagggccaa
cacgcgctgt 60 cgatcgatcg atcgatgaag ggcg 84 380 82 DNA
Artificial
clone of aptamer 380 gggagaggag agaacgttct acatatcaat agactcttga
cgtttgggtt tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 381 79 DNA
Artificial clone of aptamer 381 gggagaggag agaacgttct acagtgaagg
aaaagtaagt gaaggtgtgc gctgtcgatc 60 gatcgatcga tgaagggcg 79 382 82
DNA Artificial clone of aptamer 382 gggagaggag agaacgttct
acggatgaaa tgagtgtctg cgataggtta agcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 383 83 DNA Artificial clone of aptamer 383
gggagaggag agaacgttct acggaaggaa atgtgtgtct gcgataggtt aagcgctgtc
60 gatcgatcga tcgatgaagg gcg 83 384 82 DNA Artificial clone of
aptamer 384 gggagaggag agaacgttct acatccttgc gtatgatcgg catcgtaaga
cacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 385 82 DNA Artificial
clone of aptamer 385 gggagaggag agaacgttct acatccttgc gtatgatcgg
catcgtaaga cacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 386 77 DNA
Artificial clone of aptamer 386 gggagaggag agaacgttct acgatcgaag
tcgtgacaga aaccactcgc tgtcgatcga 60 tcgatcgatg aagggcg 77 387 77
DNA Artificial clone of aptamer 387 gggagaggag agaacgttct
acgatcgaag tcgtgacaga aaccactcgc tgtcgatcga 60 tcgatcgatg aagggcg
77 388 82 DNA Artificial clone of aptamer 388 gggagaggag agaacgttct
acggaaaagg ttggcgaaac gaagaagaat ttcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 389 82 DNA Artificial clone of aptamer 389
gggagaggag agaacgttct acggaaaagg ttggcgaaac gaagaanaat ttcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 390 83 DNA Artificial clone of
aptamer 390 gggagaggag agaacgttct actgggagtt gcggtgtttt gcggtggatt
tgacgctgtc 60 gatcgatcga tcgatgaagg gcg 83 391 83 DNA Artificial
clone of aptamer 391 gggagaggag agaacgttct actgggagtt gcggtgtttt
gcggtggatt tgacgctgtc 60 gatcgatcga tcgatgaagg gcg 83 392 82 DNA
Artificial clone of aptamer 392 gggagaggag agaacgctct acaagattgt
agatcaacag cgaaggcgtg ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 393
82 DNA Artificial clone of aptamer 393 gggagaggag agaacgctct
acaagattgt agatcaacag cgaaggcgtg ggcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 394 82 DNA Artificial clone of aptamer 394
gggagaggag agaacgttct acaaanaaga tnnccancnn gaganaaagg agcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 395 82 DNA Artificial clone of
aptamer 395 gggagaggag agaacgttct acaaacatcg aagatcgaac tgaaaaggag
ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 396 82 DNA Artificial
clone of aptamer 396 gggagaggag agaacgttct acatgtgcat gcaaggtggg
gctgacacga gccgctgtcg 60 atcgatcgat cgatgaaggg cg 82 397 80 DNA
Artificial clone of aptamer 397 gggagaggag agaacgttct acaaggagta
gatcgacaga atagaaaaat cgctgtcgat 60 cgatcgatcg atgaagggcg 80 398 83
DNA Artificial clone of aptamer 398 gggagaggag agaacgttct
acaaaaggta aggtcaaaaa agcgcaacgt tgacgctgtc 60 gatcgatcga
tcgatgaagg gcg 83 399 82 DNA Artificial clone of aptamer 399
gggagaggag agaacgttct acaaaaggag gcgaaataag tgagacaatg tgcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 400 81 DNA Artificial clone of
aptamer 400 gggagaggag agaacgttct acaaaaatcc acaaacatag ctgtaattgc
tcgctgtcga 60 tcgatcgatc gatgaagggc g 81 401 81 DNA Artificial
clone of aptamer 401 gggagaggag agacgttcta caagaacata taacattttg
gttgagagca acgctgtcga 60 tcgatcgatc gatgaagggc g 81 402 83 DNA
Artificial clone of aptamer 402 gggagaggag agaacgttct acaagagtcn
acgatttcna tcacaaatgt ggctgctgtc 60 natcgatcga tcnatgaagg gcg 83
403 83 DNA Artificial clone of aptamer 403 gggagaggag agaacgttct
acaagcaagc aaaaaaagta tcgacagaag tggcgctgtc 60 gatcgatcga
tcgatgaagg gcg 83 404 82 DNA Artificial clone of aptamer 404
gggagaggag agaacgttct acaagtaata tcagagcaat cggaataaga gtcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 405 82 DNA Artificial clone of
aptamer 405 gggagaggag agaacgttct acagacttcg atgcgatgga tttggaaatg
tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 406 82 DNA Artificial
clone of aptamer 406 gggagaggag agaacgttct acagaaagaa ttacaggaac
aaatacacgt gcggctgtcg 60 atcgatcgat cgatgaaggg cg 82 407 82 DNA
Artificial clone of aptamer 407 gggagaggag agaacgttct acagaaatca
atcgaggtga tcgttatata ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 408
82 DNA Artificial clone of aptamer 408 gggagaggag agaacgttct
acagatttgg atcgacaatc tcgtagaaga gacgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 409 82 DNA Artificial clone of aptamer 409
gggagaggag agaacgttct acaatgcaag tttaagtgtg gtgtcaaacg cacgctgtcg
60 atcgatcgat cgatgaaggg cg 82 410 81 DNA Artificial clone of
aptamer 410 gggagaggag agaacgttct acaaataaag acacgaagat cgacggagac
tcgctgtcga 60 tcgatcgatc gatgaagggc g 81 411 82 DNA Artificial
clone of aptamer 411 gggagaggag agaacgttct acgaagatgt gtttaagaat
cgaggttttc gacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 412 81 DNA
Artificial clone of aptamer 412 gggagaggag agaacgttct acgagttggc
acgcatgtat aggtattttg gcgctgtcga 60 tcgatcgatc gatgaagggc g 81 413
84 DNA Artificial clone of aptamer 413 gggagaggag agaacgttct
acgaaaaaaa gagatgagag aaaggattaa gagacgctgt 60 cgatcgatcg
atcgatgaag ggcg 84 414 82 DNA Artificial clone of aptamer 414
gggagaggag agaacgttct acgaaaagga aaaaaaacga tcggcagagt cccgctgtcg
60 atcgatcgat cgatgaaggg cg 82 415 82 DNA Artificial clone of
aptamer 415 gggagaggag agaacgttct acgattaagg aaacatttac gcgaatacat
gacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 416 81 DNA Artificial
clone of aptamer 416 gggagaggag agaacgttct acgacgtttg ctctgaaaat
aggacagaag gcgctgtcga 60 tcgatcgatc gatgaagggc g 81 417 82 DNA
Artificial clone of aptamer 417 gggagaggag agaacgttct acgaagatgt
gtttaagaat cgaggttttc gacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 418
82 DNA Artificial clone of aptamer 418 gggagaggag agaacgttct
accgagatcg aaaggtaaga gaaaattcat ggcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 419 82 DNA Artificial clone of aptamer 419
gggagaggag agaacgttct actaagattc gtcgttcaga cagagaaagc gacgctgtcg
60 atcgatcgat cgatgaaggg cg 82 420 84 DNA Artificial clone of
aptamer 420 gggagaggag agaacgttct accttggcga cgatctgtga cctgaatttt
tgtccgctgt 60 cgatcgatcg atcgatgaag ggcg 84 421 84 DNA Artificial
clone of aptamer 421 gggagaggag agaacgttct accttggcga cgatctgtga
cctgaatttt tgtccgctgt 60 cgatcgatcg atcgatgaag ggcg 84 422 83 DNA
Artificial clone of aptamer 422 gggagaggag agaacgttct accttggtct
cagcagcttt taacaaagta tcccgctgtc 60 gatcgatcga tcgatgaagg gcg 83
423 83 DNA Artificial clone of aptamer 423 gggagaggag agaacgttct
accttggtct cagcagcttt taacaaagta tcccgctgtc 60 gatcgatcga
tcgatgaagg gcg 83 424 81 DNA Artificial clone of aptamer 424
gggagaggag agaacgttct accgctattt tgttcattga aggacttgtc acgctgtcga
60 tcgatcgatc gatgaagggc g 81 425 81 DNA Artificial clone of
aptamer 425 gggagaggag agaacgttct accgctattt tgttcattga aggacttgtc
acgctgtcga 60 tcgatcgatc gatgaagggc g 81 426 82 DNA Artificial
clone of aptamer 426 gggagaggag agaacgttct accctattga ggttgattgg
aagtgcctat gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 427 82 DNA
Artificial clone of aptamer 427 gggagaggag agaacgttct accctattga
ggttgattgg aagtgcctat gtcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 428
81 DNA Artificial clone of aptamer 428 gggagaggag agaacgttct
actgaagatg ttatgatgat tgacgaggag gcgctgtcga 60 tcgatcgatc
gatgaagggc g 81 429 81 DNA Artificial clone of aptamer 429
gggagaggag agaacgttct actgaagatg ttatgatgat tgacgaggag gcgctgtcga
60 tcgatcgatc gatgaagggc g 81 430 82 DNA Artificial clone of
aptamer 430 gggagaggag agaacgttct actgtctgag tgtcgccgcc ttgtgtgatg
ttcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 431 82 DNA Artificial
clone of aptamer 431 gggagaggag agaacgttct actgtctgag tgtcgccgcc
ttgtgtgatg ttcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 432 82 DNA
Artificial clone of aptamer 432 gggagaggag agaacgttct acgtgatggc
tgtgaatgag gtagttcgaa tacgctgtcg 60 atcgatcgat cgatgaaggg cg 82 433
81 DNA Artificial clone of aptamer 433 gggagaggag agaacgttct
acgtgaaatc aaggttgtta atttggggaa tcgctgtcga 60 tcgatcgatc
gatgaagggc g 81 434 82 DNA Artificial clone of aptamer 434
gggagaggag agaacgttct acgtataagg ccgtaaccgg gtagcgagtg gtcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 435 82 DNA Artificial clone of
aptamer 435 gggagaggag agaacgttnt acgtgggcga aggagctgcg ggcgttgnag
tttgctgtcg 60 atcgatcgat cgatgaaggg cg 82 436 82 DNA Artificial
clone of aptamer 436 gggagaggag agaacgttct acgtcatcct agtctgagat
cggattttct tgcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 437 82 DNA
Artificial clone of aptamer 437 gggagaggag agaacgttct acgtttgcga
gtgtggtcga cgctgaatgc ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 438
82 DNA Artificial clone of aptamer 438 gggagaggag agaacgttct
acggattgat agggattgag atgaggtctt gtcgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 439 81 DNA Artificial clone of aptamer 439
gggagaggag agaacgttct acgatgtcgt gttagattac ttattgctat ctgctgtcga
60 tcgatcgatc gatgaagggc g 81 440 82 DNA Artificial clone of
aptamer 440 gggagaggag agaacgttct acgatgcctg gcggaaacgg agcctgggat
ttcgctgtcn 60 atcgatcgat cgatgaaggg cg 82 441 80 DNA Artificial
clone of aptamer 441 gggagaggag agaacgttct acgaggattt gacgtgtgtg
tgctagagta cgctgtcgat 60 cgatcgatcg atgaagggcg 80 442 82 DNA
Artificial clone of aptamer 442 gggagaggag agaacgttct acgagtatta
tgcgtccctt gaggatacac ggcgctgtcg 60 atcgatcgat cgatgaaggg cg 82 443
82 DNA Artificial clone of aptamer 443 gggagaggag agaacgttct
acagggataa ctgtagcgat gaaagtaaac gatgctgtcg 60 atcgatcgat
cgatgaaggg cg 82 444 81 DNA Artificial clone of aptamer 444
gggagaggag agaacgttct acaagaagtg tggccgcaga gacgaaatgc acgctgtcga
60 tcgatcgatc gatgaagggc g 81 445 83 DNA Artificial clone of
aptamer 445 gggagaggag agaacgttct acccatatct tccttcttta ttccgttagt
tgccgctgtc 60 gatcgatcga tcgatgaagg gcg 83 446 82 DNA Artificial
clone of aptamer 446 gggagaggag agaacgttct acctgtgttg atgcttccgt
ttgagattgc cccgctgtcg 60 atcgatcgat cgatgaaggg cg 82 447 84 DNA
Artificial clone of aptamer 447 gggagaggag agaacgttct accngtaaga
naanctattt tagcccttgn nctgcgctgt 60 cgatcgatcg atcgatgaag ggcg 84
448 83 DNA Artificial clone of aptamer 448 gggagaggag agaacgttct
acccttgtcc tccaatcctc ttttgactct tgccgctgtc 60 gatcgatcga
tcgatgaagg gcg 83 449 82 DNA Artificial clone of aptamer 449
gggagaggag agaacgttct acctgatttt gtcactggat tccgatggct ttcgctgtcg
60 atcgatcgat cgatgaaggg cg 82 450 83 DNA Artificial clone of
aptamer 450 gggagaggag agaacgttct actgtaataa gggatgcgtc aggaacctgt
gttcgctgtc 60 gatcgatcga tcgatgaagg gcg 83 451 81 DNA Artificial
clone of aptamer 451 gggagaggag agaacgttct actgctttcc gggaatttgt
ttgtttgctt ccgctgtcga 60 tcgatcgatc gatgaagggc g 81 452 82 DNA
Artificial clone of aptamer 452 gggagaggag agaacgttct acttcgtcgg
ttgacttttc ttcgtgtagt gtcgctgtcg 60 atcgattgat cgatgaaggg cg 82 453
92 DNA Artificial aptamer library template 453 catcgatcga
tcgatcgaca gcgnnnnnnn nnnnnnnnnn nnnnnnnnnn nnngtagaac 60
gttctctcct ctccctatag tgagtcgtat ta 92 454 24 DNA Artificial PCR
3'-primer 454 catcgatgct agtcgtaacg atcc 24 455 40 DNA Artificial
PCR 5'-primer 455 taatacgact cactataggg agaggagaga aacgttctcg 40
456 75 RNA Artificial rRmY aptamer ARC256 RNA transcription product
456 gggagaggag agaacguucu acnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nncgcugucg 60 aucgaucgau cgaug 75 457 11 RNA Artificial mN PEG 5'
oligonucleotide 457 ggngcngcnc c 11 458 19 RNA Artificial mN PEG 3'
oligonucleotide 458 ggugccnngu cguugcucc 19 459 75 DNA Artificial
aptamer library template 459 gggagaggag agaacgttct acnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nncgctgtcg 60 atcgatcgat cgatg 75 460 22 DNA
Artificial PCR primer 460 gggagaggag agaacgttct ac 22 461 22 DNA
Artificial PCR primer 461 catcgatcga tcgatcgaca gc 22 462 75 RNA
Artificial rGmH aptamer ARC256 transcription product 462 gggngnggng
ngnncguucu ncnnnnnnnn nnnnnnnnnn nnnnnnnnnn nncgcugucg 60
nucgnucgnu cgnug 75 463 75 RNA Artificial r/mGmH aptamer ARC256
transcription product 463 gggngnggng ngnncguucu ncnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nncgcugucg 60 nucgnucgnu cgnug 75 464 75 DNA
Artificial dRmY aptamer ARC256 transcription product 464 gggagaggag
agaacguucu acnnnnnnnn nnnnnnnnnn nnnnnnnnnn nncgcugucg 60
aucgaucgau cgaug 75 465 11 DNA Artificial dRmY PEG 5'
oligonucleotide 465 ggagcagcac c 11 466 19 DNA Artificial dRmY PEG
3' oligonucleotide 466 ggugccaagu cguugcucc 19 467 80 DNA
Artificial clone of aptamer 467 gggagaggag agaacgttct acttgctgtg
acggacgggc ttgagaggct cgctgtcgat 60 cgatcgatcg atgaagggcg 80 468 75
RNA Artificial rN aptamer ARC256 transcription product 468
gggagaggag agaacguucu acnnnnnnnn nnnnnnnnnn nnnnnnnnnn nncgcugucg
60 aucgaucgau cgaug 75
* * * * *